Methods for the manufacture of proteolytically processed polypeptides

ABSTRACT

The present invention relates to a novel proteolytically active polypeptide and various uses of the polypeptide (and others) in screening and manufacturing methods.

This application is a national stage application of International PatentApplication No. PCT/EP2012/073283, filed on Nov. 21, 2012, pending.

Pursuant to the provisions of 37 C.F.R. § 1.52(e)(5), the sequencelisting text file named 103355_Seq_Lstng.txt, created on Feb. 19, 2015and having a size of 29,619 bytes, and which is being submittedherewith, is incorporated by reference herein in its entirety.

The present invention relates to a novel proteolytically activepolypeptide and various uses of the polypeptide in screening andmanufacturing methods.

Clostridium botulinum and Clostridium tetani produce highly potentneurotoxins, i.e. botulinum neurotoxins (BoNTs) and tetanus neurotoxin(TeNT), respectively. These clostridial neurotoxins (CNTs) specificallybind to neuronal cells and disrupt neurotransmitter release. Clostridiumbotulinum secretes seven antigenically distinct serotypes designated Ato G of the botulinum neurotoxin (BoNT). All serotypes together with therelated tetanus neurotoxin (TeNT) secreted by Clostridium tetani, areZn²⁺-endoproteases that block synaptic exocytosis by cleaving proteinsinvolved in the formation of the SNARE complex controlling cellularmembrane fusion. CNTs cause the flaccid muscular paralysis seen inbotulism and tetanus. In addition, CNT activity has been shown to affectglandular secretion. These physiological effects of CNTs on muscular andglandular activity are increasingly used in various therapeutic andcosmetic applications. Botulinum neurotoxin of serotype A (BoNT/A) wasapproved for human use in the United States in 1989 for the treatment ofstrabism, blepharospasm, and other disorders. It is commerciallyavailable as a botulinum neurotoxin A protein preparation, for example,under the tradename BOTOX (Allergan Inc.) and under the tradenameDYSPORT (Ipsen Ltd). For therapeutic application, a complex comprisingthe neurotoxin and additional bacterial proteins is injected directlyinto the muscle to be treated. At physiological pH, the toxin isreleased from the protein complex (Eisele et al. 2011, Toxicon57(4):555-65.) and the desired pharmacological effect takes place. Animproved BoNT/A preparation being free of complexing proteins isavailable under the tradenames XEOMIN or Bocouture (Merz PharmaceuticalsGmbH, Frankfurt/Germany). The effect of BoNT is only temporary, which isthe reason why repeated administration of BoNT is usually required tomaintain a therapeutic affect.

Each CNT is initially synthesised as an inactive single chainpolypeptide. In the case of BoNT, the neurotoxin polypeptide has amolecular weight of approximately 150 kDa. The posttranslationalprocessing of this single chain polypeptide involves limited proteolysisin an exposed region called loop (see table 1) and the formation of anearby disulfide bridge. Active di-chain neurotoxin consists of twocleavage products resulting from the proteolytic hydrolysis of thesingle chain precursor polypeptide: an N-terminal light chain of approx.50 kDa and a heavy chain of approx. 100 kDa linked by a disulfide bond.CNTs structurally consist of three domains, i.e. the catalytic lightchain, the heavy chain encompassing the translocation domain (N-terminalhalf) and the receptor binding domain (C-terminal half) (cf. Krieglstein1990, Eur J Biochem 188: 39; Krieglstein 1991, Eur J Biochem 202: 41;Krieglstein 1994, J Protein Chem 13: 49; Lacy et al., 1998, Nat. Struct.Biol. 5(10):898-902). Depending on the number of cleavage sites presentin the single chain between the amino acid residues forming thecatalytic domain and the amino acid residues forming the translocationdomain, endopeptidase activity can give rise to two large cleavageproducts, i.e. the light and the heavy chain and, in addition,characteristic short peptides representing the former loop region,bridging in the single chain of the neurotoxin what is to become thelight and the heavy chain (cf. table 1, below).

The purification of the CNTs from the fermentation solution is aparticular challenge, since the neurotoxins are contained therein as amixture of unprocessed, partially processed and fully processedpolypeptides, all of which have very similar biochemical and physicalproperties. Partially processed neurotoxins are typically generated, ifthe endoproteolytic activity has hydrolysed the peptide bond between thelight chain and the loop, while the peptide bond between the loop andthe N-terminus of the heavy chain is still intact. Moreover, partiallyprocessed neurotoxin can also be created if the endoproteolytic activityhas released the loop peptide from the heavy chain, while the peptidebond between the loop peptide and the C-terminus of the light chain hasnot yet been hydrolysed. Depending on the conditions of fermentation andthe type of neurotoxin, the fully processed polypeptide which is devoidof the loop peptide can be contaminated significantly, with between 5%to 90% partially processed or unprocessed polypeptide. Yet in somecases, the neurotoxin is mainly unprocessed and, prior to therapeuticuse, needs to be treated with an endopeptidase in order to becomebiologically active.

The prior art describes various attempts to treat clostridialneurotoxins with heterologous proteases in order to reduce the amount ofunprocessed or partially processed precursor protein. The protease mostwidely used for activation of clostridial neurotoxins, Trypsin, whilebeing useful for activating clostridial neurotoxins of serotypes B(BoNT/B) and E (BoNT/E) (DasGupta & Sugiyama 1972, Biochem. Biophys.Res. Commun. 48: 108-112; Kozaki et al., 1974, Infect. Immun. 10:750-756) appears to produce secondary products, presumably byproteolytic action near the C-terminus of the heavy subunit of BoNT/Aand, thus, appears to destroy toxin binding to its cellular receptor(Shone et al., 1985, Eur. J. Bioch. 151: 75-82). More specific cleavageproducts are theoretically expected from endogenous proteases, isolatedfrom the native host, such as C. botulinum producing BoNT/A.Accordingly, various attempts have been made to isolate from the nativehost cell the endogenous protease involved in proteolytic activation ofclostridial neurotoxins. Dekleva & DasGupta (Dekleva & DasGupta, 1989,Biochem. Biophys. Res. Commun. 162: 767-772) purified from cultures ofC. botulinum producing BoNT/A a fraction capable of proteolyticallycleaving BoNT/A into a heavy and a light subunit. Later studies of thesame authors further characterised the endogenous protease isolated fromC. botulinum (Dekleva & DasGupta, 1990, J. Bact. 172: 2498-2503) andrevealed a 62 kDa protein, composed of a 15.5 kDa polypeptide and a 48kDa polypeptide. However, the observation of considerable fragmentationof CNTs after limited exposure to the 62 kDa protein of Dekleva &DasGupta suggests that the isolated protease may not be the unidentifiedproteolytic enzyme responsible for the activation of CNTs in clostridialcell cultures and during infection. In fact, others have recentlysuggested that Clostripain, also designated clostridiopeptidase B(Mitchel & Harrington, 1968, JBC 243: 4683-4692), might be involved inthe specific activation of CNTs (Sebaihia et al., 2007, Genome Res.17(7):1082-1092; WO2009/014854). Interestingly, the structure andsubstrate specificity of this enzyme are reminiscent of those of thesecreted alpha-clostripain from Clostridium histolyticum (Dargatz et al.1993), a homolog (74% amino acid identity) of which is present in C.botulinum (CBO1920). The C. histolyticum alpha-clostripain is a cysteineendopeptidase with strict specificity for the arginyl bond. It issynthesized as an inactive prepro-enzyme that undergoes an autocatalyticcleavage to generate 15.4- and 43-kDa polypeptides, which associate toform a heterodimeric active enzyme (Dargatz et al. 1993). Both the C.histolyticum alphaclostripain and the C. botulinum 62-kDa proteaserequire a reducing agent and calcium for full activity and aresusceptible to the same protease inhibitors. These data strongly suggestthat the C. botulinum ortholog of alpha-clostripain (CBO1920) is theendogenous protease responsible for the proteolytic nicking of theneurotoxin of C. botulinum. A gene encoding clostripain (CPE0846) isalso present in C. perfringens, and has been found to be positivelyregulated by the two-component system VirR/VirS (Shimizu et al. 2002b).

Till this day, however, further conclusive experimental evidence ismissing and a protease capable of efficiently converting the singlechain precursor CNTs into the authentic mature cleavage products, i.e.the di-chain neurotoxin, is still not available in the art. The presentinvention solves one or more of the above-described problems.

Means and methods for reducing the amount of unprocessed and/orpartially processed neurotoxin polypeptides and thereby improving thequality of neurotoxin preparations are highly desirable but not yetavailable. Thus, a technical problem underlying the present inventionmay be seen as the provision of means and methods for improving themanufacture of neurotoxin polypeptides by complying with theaforementioned needs. The technical problem is solved by the embodimentscharacterised in the claims and herein below.

Accordingly, the present invention relates in one aspect to aproteolytically active polypeptide which comprises a polypeptidesequence having at least 50% sequence identity with the sequence of SEQID NO: 1. In another aspect, the present invention relates to aproteolytically active polypeptide consisting of a polypeptide sequencehaving at least 50% sequence identity with the sequence of SEQ ID NO: 1.In another aspect, the present invention relates to a proteolyticallyactive polypeptide consisting of a polypeptide sequence as shown in SEQID NO: 1.

The term “proteolytically active polypeptide” as used herein refers tothe catalytic function of the polypeptide of the present invention andmeans that the polypeptide of the present invention is capable ofhydrolysing a peptide bond. In one aspect, “proteolytically activepolypeptide” refers to a polypeptide that is capable of hydrolysing apolypeptide comprising an amino acid sequence selected from any one ofSEQ ID NOs: 4 to 25. The term “proteolytically inactive polypeptide” asused herein refers to the catalytic function of the polypeptide of thepresent invention and means that the polypeptide of the presentinvention is incapable of hydrolysing a peptide bond.

The skilled person can determine whether a polypeptide according to thesequence definition mentioned herein is a polypeptide according to thepresent invention, by testing the proteolytic activity of saidpolypeptide. An assay or test system for determining proteolyticactivity comprises contacting a polypeptide which comprises apolypeptide sequence having at least 50% sequence identity with thesequence of SEQ ID NO: 1 with a test substrate. A test substrate istypically a polypeptide which is known to be cleavable by thepolypeptide of the present invention. Preferably, the test substrate isa CNT such as BoNT or a fragment thereof. The test substrate can be e.g.uncleaved/unprocessed BoNT, designated herein as “scBoNT” and can bee.g. of serotype A, B, C1, D, E, F or G (e.g. “scBoNT/A”, “scBoNT/B”etc.) or the test substrate can be tetanus neurotoxin. Alternatively,the test substrate can be a fragment of a clostridial neurotoxin, saidfragment comprising an amino acid sequence selected from any one of SEQID NOs: 4 to 25. The fragment can be a polypeptide of 50 or more aminoacid residues or a peptide of up to 49 amino acid residues. As usedthroughout the present specification, the term “polypeptide” refers tomolecules with 50 or more amino acid residues, whereas the term“peptide” refers to molecules with 2 to 49 amino acid residues. In oneaspect, the test substrate is a soluble neurotoxin fragment calledLH_(N) comprising the light chain polypeptide, the exposed loop peptideregion and the N-terminal half of the heavy chain polypeptide, thetranslocation domain H_(N). In another aspect, the test substrate is orcomprises a peptide selected from any one of SEQ ID NOs: 4 to 25 (cf.table 1). In yet another aspect, the test substrate is a chimericneurotoxin comprising amino acid residues derived from two or moreserotypes.

An assay for determining the proteolytic activity would typicallycomprise a step of determining the degree of conversion of the testsubstrate into its cleavage product(s). The observation of one or morecleavage product(s) generated after contacting the polypeptide with thetest substrate or the observation of an increase in the amount ofcleavage product(s) is indicative of proteolytic activity of thepolypeptide. Said step of determining may involve comparing substrateand cleavage product(s). Said comparing may involve determining theamount of substrate and/or the amount of one or more cleavage product(s)and may also involve calculating the ratio of substrate and cleavageproduct(s). In addition, the assay for determining the proteolyticactivity may comprise a step of comparing a test sample with a referencesample, wherein the reference sample typically comprises (a) apolypeptide which comprises a polypeptide sequence having at least 50%sequence identity with the sequence of SEQ ID NO: 1 and which is knownto be proteolytically active and (b) a test substrate known to becleavable by the polypeptide of (a). In one aspect, the assay fordetermining the proteolytic activity comprises separating substrate andcleavage product(s) by electrophoresis or by column chromatography and,optionally, a spectrometric analysis. It may be convenient to label thetest substrate with one or more labels in order to more easily detectdecrease of test substrate and/or increase of product(s). The term“label”, as used herein, means detectable marker and includes e.g.radioactive label, antibody, fluorescent label. The amount of testsubstrate and/or cleavage product may be determined e.g. by methods ofautoradiography or spectrometry, including methods based on energyresonance transfer between at least two labels. Alternatively,immunological methods such as western blot or ELISA may be used fordetection. A preferred assay for determining the proteolytic activity ofthe polypeptide of the present invention is described herein below inthe Examples illustrating the invention. In a particularly preferredembodiment of the present invention, a polypeptide is proteolyticallyactive, if more than 20%, preferably more than 95% of test substrate isconverted into the cleavage products such as the light chain and theheavy chain in 120 min at 37° C. using a buffer selected from 100 mMTris-HCl, pH 8.0 or PBS (50 mM Na₂HPO₄, 150 mM NaCl, pH 7.4). The sameconditions apply, if the test substrate is not a full length neurotoxinbut, instead, e.g. a fragment of the full length neurotoxin or aderivative of the neurotoxin. It is apparent that the cleavage productswill differ in this case. However, the skilled person can quantify thecorresponding cleavage products. In another aspect, typically, 100 ng ofproteolytically active polypeptide and a molar ratio of 1:100 withregard to the substrate are used in the assay. In yet another aspect, asample may be taken at intervals in order to follow the catalyticactivity over time. The assay may be modified, e.g. by using multipleamounts of the proteolytically active polypeptide.

SEQ ID NO: 2 shows the polypeptide sequence of a proteolyticallyinactive polypeptide derived from a Clostridium botulinum strain ATCC3502, GenBank accession No: “CAL82988.1”, having an amino acid length of581 residues. SEQ ID NO: 1 shows a proteolytically active derivative ofSEQ ID NO: 2, lacking amino acid residues 1 to 248 of SEQ ID NO: 2.

The term “polypeptide which comprises a polypeptide sequence having atleast 50% sequence identity with the sequence of SEQ ID NO: 1” refers toa polypeptide which has at least 50% sequence identity with the sequenceof SEQ ID NO: 1. In addition, the term refers to a polypeptide whichcomprises a polypeptide sequence having at least 50% sequence identitywith the sequence of SEQ ID NO: 1. Said polypeptide may have additionalamino acids, for example at an internal position or N- or C-terminal tothe sequence shown in SEQ ID NO: 1 or at an internal position or N- orC-terminal to a amino acid sequence which is at least 50% identical withsequence of SEQ ID NO: 1, wherein a methionine may be present at theN-terminus of the polypeptide. In addition, the term refers to apolypeptide lacking one or more amino acid residues, for example at aninternal position or at the N- or C-terminus of the sequence shown inSEQ ID NO: 1 or at an internal position or the N- or C-terminus of asequence which is at least 50% identical in sequence to SEQ ID NO: 1.

The term “sequence identity” as used herein refers to determination ofthe identity between a reference amino acid sequence and a querysequence wherein the sequences are aligned so that the highest ordermatch is obtained, and which can be calculated using publishedtechniques or methods codified in computer programs such as, forexample, BLASTP, BLASTN, FASTA (Altschul 1990, J Mol Biol 215: 403). Thepercent identity values are in one aspect calculated over the entireamino acid sequence. In another aspect, sequence identity is calculatedover a sequence length of up to 50aa residues, up to 100aa, up to 150aa,up to 250aa, 300aa, 350aa, 400aa, 450aa, 500aa, or 550aa residues. Inanother aspect, sequence identity is calculated over at least 50aaresidues, at least 100aa, at least 150aa or at least 250aa residues. Inpreferred embodiments, sequence identity is determined over the entirelength of SEQ ID NO: 1 or 2, i.e. over a length of 333aa or 581aa,respectively. A series of programs based on a variety of algorithms isavailable to the skilled worker for comparing different sequences. Inthis context, the algorithms of Needleman and Wunsch or Smith andWaterman give particularly reliable results. To carry out the sequencealignments and calculate the sequence identity values recited herein,the commercially available program DNASTAR Lasergene MegAlign version7.1.0 based on the algorithm Clustal W was used over the entire sequenceregion with the following settings: Pairwise Alignment parameters: GapPenalty: 10.00, Gap Length Penalty: 0.10, Protein weight matrix Gonnet250, which, unless otherwise specified, shall always be used as standardsettings for sequence alignments.

The term “at least 50% sequence identity” as used herein means at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%.

The proteolytically active polypeptide of the present invention may havethe same number of amino acids as the reference polypeptide sequence asshown in SEQ ID NO: 1. Also comprised by the present invention arepolypeptides having additional or less amino acid residues. In oneaspect, the proteolytically active polypeptide of the present inventionis or comprises a truncation mutant of SEQ ID NO:1 or 2 or of apolypeptide having at least 50% sequence identity with the sequence ofSEQ ID NO: 1 or 2. The truncation mutant of SEQ ID NO: 2 may for examplelack one or more amino acid residues N-terminal to amino acid position249. A truncation mutant may be an N- or C-terminal truncation mutantand/or an internal truncation mutant that is proteolytically active. Inone aspect, said truncation mutant of SEQ ID NO:2 lacks amino acidpositions 1 to 248 of SEQ ID NO: 2. In another aspect, the truncationmutant of SEQ ID NO: 2 is a C-terminal truncation mutant. In one aspect,the truncation mutant lacks up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,50, 100, 150 or up to 170 consecutive amino acid residues. In anotheraspect, the proteolytically active polypeptide of the present inventionhas an amino acid length of at least 200aa residues, of at least 250aaresidues, of at least 300aa residues or of at least 333aa residues. Inanother aspect, the proteolytically active polypeptide of the presentinvention has up to 333aa residues, up to 350aa residues, up to 573residues, up to 581aa residues, up to 592aa residues, up to 600aa or upto 617aa residues.

In another aspect, the proteolytically active polypeptide of the presentinvention encompasses a polypeptide comprising additional amino acidresidues at the N- or C-terminus and/or at an internal position of thepolypeptide chain of SEQ ID NO: 1 or a of a polypeptide sequence havingat least 50% sequence identity with the sequence of SEQ ID NO: 1. Theseadditional amino acid residues may comprise up to 5, up to 10 or even upto 200, 300 or up to 400 consecutive amino acid residues. In one aspect,the additional amino acid residues function as an inhibitor of theproteolytic activity. In another aspect, the additional amino acidresidues can be removed by a protease. In another aspect, additionalresidues inhibiting the proteolytic activity of the polypeptide of thepresent invention are excluded. The additional amino acid residues maybe flanked by one or more protease cleavage sites. In another aspect,the additional amino acid sequence functions as a detectable tag and/orallows binding to a solid support.

In another aspect, the polypeptide chain of SEQ ID NO: 1 or a of apolypeptide sequence having at least 50% sequence identity with thesequence of SEQ ID NO: 1 is modified by exchanging one or more aminoacid residues. The term “exchanging”, as used herein, means replacing anamino acid with a different amino acid. For example, up to 1 aa, 2aa,3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 15aa, 20aa or up to 50aa may bereplaced within the polypeptide sequence. The exchanges may involveconservative or non-conservative amino acid changes, aiming e.g. atincreasing or decreasing substrate binding or proteolytic activity ofthe polypeptide of the present invention.

In one aspect, the proteolytically active polypeptide of the presentinvention encompasses a polypeptide that is capable of hydrolysing asubstrate into two or more native cleavage product(s). In anotheraspect, the polypeptide of the present invention hydrolyses thesubstrate into two or more cleavage products which differ from thenative cleavage products. The term “native cleavage products” or “nativeproducts” as used herein refers to products, which are identical inamino acid sequence when compared to products generated from the samesubstrate in wildtype cell cultures, from which the substrateoriginates. In one aspect the cleavage product is the di-chainneurotoxin of a botulinum neurotoxin or of tetanus neurotoxin, inanother aspect the di-chain neurotoxin is a neurotoxin isolated from C.botulinum of serotype A, B, C1, D, E, F or G. In yet another aspect,said di-chain neurotoxin is a native di-chain neurotoxin.

Table 1 shows the precursor, the native di-chain neurotoxin of TeNT andof BoNT/A-G and identifies the exposed loop comprising the amino acidsequence cleaved by the polypeptide of the present invention.

Toxin exposed loop LC H_(N) H_(CN) H_(CC) BoNT/A1 SEQ ID NO: 4 M1-K438A449-N872 I873-S1092 N1093-L1296 BoNT/A2 SEQ ID NO: 5 M1-K438 A449-N872I873-S1092 N1093-L1296 BoNT/A3 SEQ ID NO: 6 M1-K434 A445-N868 I869-S1088N1089-L1292 BoNT/A3 SEQ ID NO: 7 M1-K434 A445-N868 I869-S1088N1089-L1292 BoNT/A4 SEQ ID NO: 8 M1-K438 A449-N872 I873-S1092N1093-L1296 BoNT/A5 SEQ ID NO: 9 M1-K438 A449-N872 I873-S1092N1093-L1296 BoNT/A6 SEQ ID NO: 5 M1-K438 A449-N872 I873-S1093N1094-L1297 BoNT/A7 SEQ ID NO: 10 M1-K438 A449-N872 I873-S1092N1093-L1296 BoNT/B1 SEQ ID NO: 11 M1-K441 A442-I860 L861-S1079Y1080-E1291 BoNT/B2 SEQ ID NO: 12 M1-R441 A442-I860 L861-S1079Y1080-E1291 BoNT/B3 SEQ ID NO: 12 M1-R441 A442-I860 L861-S1079Y1080-E1291 BoNT/B4bv SEQ ID NO: 11 M1-K441 A442-I860 L861-S1079Y1080-E1291 BoNT/B5nP SEQ ID NO: 13 M1-K441 V442-I860 L861-S1079Y1080-E1291 BoNT/B6 SEQ ID NO: 11 M1-K441 A442-I860 L861-S1079Y1080-E1291 BoNT/C1 SEQ ID NO: 14 M1-R444 T450-I868 N869-L1092Q1093-E1291 BoNT/CD SEQ ID NO: 14 M1-R444 T450-I868 N869-Q1083I1084-E1280 BoNT/D SEQ ID NO: 15 M1-K442 D446-I864 N865-Q1079I1080-E1276 BoNT/DC SEQ ID NO: 16 M1-R442 D446-I864 N865-L1088Q1089-E1285 BoNT/E1-E5 SEQ ID NO: 17 M1-K419 S424-I847 K848-P1067N1068-K1252 BoNT/E6 SEQ ID NO: 18 M1-K419 S424-I847 K848-P1067N1068-K1252 BoNT/F1 SEQ ID NO: 19 M1-R435 A440-I866 K867-P1085D1086-N1278 BoNT/F2 SEQ ID NO: 20 M1-R435 Q440-I866 K867-P1088D1089-E1280 BoNT/F3 SEQ ID NO: 20 M1-R435 Q440-I866 K867-P1088D1089-E1279 BoNT/F4 SEQ ID NO: 21 M1-R435 A440-I866 K867-P1085D1086-E1277 BoNT/F5 SEQ ID NO: 22 M1-K434 P440-I863 K864-P1085D1086-E1277 BoNT/F6 SEQ ID NO: 19 M1-R435 A440-I866 K867-P1088D1089-E1275 BoNT/F7 SEQ ID NO: 23 M1-K427 N432-I857 I858-P1076D1077-E1268 BoNT/G SEQ ID NO: 24 M1-K442 S447-I865 S866-S1086S1087-E1297 TeNT SEQ ID NO: 25 M1-R449 T456-K883 S884-L1109 S1110-D1315

It is to be understood that the definitions and explanations of theterms made above and below apply mutatis mutandis for all aspectsdescribed in this specification unless otherwise indicated.

The proteolytically active polypeptide of the present invention issuitable for various applications. A commercially relevant applicationis its use in the production of therapeutic neurotoxins, such as thoseisolated from C. botulinum. At present, the cell cultures of C.botulinum used for the preparation of commercially availablepreparations of botulinum neurotoxin are contaminated with significantamounts of partially processed and/or unprocessed neurotoxin, both ofwhich negatively impair, i.e. reduce the specific activity of thesepharmaceutical compositions. Using the proteolytically active oractivated polypeptide of the present invention for example after lysisof C. botulinum, it will now be possible to treat compositionscomprising unprocessed and/or partially processed neurotoxin and, thus,convert these contaminants into fully processed neurotoxin. Inconsequence, commercial products can be provided with an increasedspecific activity of the neurotoxin, wherein the total amount ofbacterial protein can be reduced, further reducing the patients risk ofantibody formation.

In another aspect, the present invention relates to a nucleic acidmolecule comprising a nucleic acid sequence encoding the polypeptide ofthe present invention and, optionally, regulatory elements. The term“regulatory elements” as used herein refers to regulatory elements ofgene expression, including transcription and translation, and includeselements such as tata box, promotor, enhancer, ribosome binding site,Shine-Dalgarno-sequence, IRES-region, polyadenylation signal, terminalcapping structure, and the like. Said regulatory element may compriseone or more heterologous regulatory elements or one or more homologousregulatory elements. A “homologous regulatory element” is a regulatoryelement of a wildtype cell, from which the nucleic acid molecule of thepresent invention is derived, which is involved in the regulation ofgene expression of the nucleic acid molecule or the polypeptide in saidwildtype cell. The present invention also encompasses nucleic acidmolecules comprising heterologous regulatory elements. The term“heterologous regulatory element” is a regulatory element which is notinvolved in the regulation of gene expression of the nucleic acidmolecule or the polypeptide in said wildtype cell. Also regulatoryelements for inducible expression, such as inducible promoters, areencompassed. The nucleic acid molecule can be, for example, hnRNA, mRNA,RNA, DNA, PNA, LNA, and/or modified nucleic acid molecules. The nucleicacid molecule can be circular, linear, integrated into a genome orepisomal. Also concatemers coding for fusion proteins comprising three,four, five, six, seven, eight, nine or ten polypeptides of the presentinvention are encompassed. Moreover, the nucleic acid molecule maycontain sequences encoding signal sequences for intracellular transportsuch as signals for transport into an intracellular compartment or fortransport across the cellular membrane.

In another aspect, the present invention relates to a vector comprisinga nucleic acid molecule according to the nucleic acid molecule of thepresent invention. A vector may be suitable for in vitro and/or in vivoexpression of the polypeptide of the present invention. The vector canbe a vector for transient and/or stable gene expression. In oneembodiment the vector furthermore comprises regulatory elements and/orselection markers. Said vector in one embodiment is of virus origin, inanother embodiment of phage origin, in yet another embodiment ofbacterial origin.

In another aspect, the present invention relates to a cell comprisingthe nucleic acid molecule or the vector of the present invention. Theterm “cell” as used herein, encompasses prokaryotic and/or eukaryoticcells suitable to express said nucleic acid molecule or said vector andin particular the polypeptide of the invention. Said cell may be a hostcell not expressing the polypeptide of the present invention or ahomolog thereof. The term “homolog” as used herein refers to apolypeptide comprising a polypeptide sequence having at least 50%sequence identity with the sequence of SEQ ID NO: 1. However, alsoencompassed by the present invention are cells, in particular wildtypecells, expressing the polypeptide of the present invention or a homologthereof. In a particular aspect, the cell of the present invention isselected from C. botulinum, C. butyricum, C. baratii and C. tetani. In apreferred aspect, the cell is C. botulinum of serotype A, B or F. Inanother aspect, said cell is the Hall strain (ATCC 3502) of C.botulinum. In another aspect, said cell is the BoNT/A producing strainATCC 19397 also known as NCTC 4587 and NCTC 7272 of C. botulinum. Inanother aspect, said cell is the BoNT/A producing strain NCTC 2916 of C.botulinum. In another aspect, said cell are the BoNT/A2 producingstrains Kyoto-F and Mauritius/NCTC 9837 of C. botulinum. In anotheraspect, said cell is the BoNT/A3 producing strain A254 Loch Maree/NCTC2012 of C. botulinum. In another aspect, said cell is the BoNT/A4 and Bproducing strain CDC657 of C. botulinum. In another aspect, said cell isthe BoNT/A5 and B3′ producing strain H04402 065 of C. botulinum. Inanother aspect, said cell is the BoNT/B1 producing strain Okra/NCTC 7273of C. botulinum. In another aspect, said cell is the BoNT/B and Fproducing strain CDC4013/NCTC 12265 of C. botulinum. In another aspect,said cell is the BoNT/F1 producing strain Langeland/NCTC 10281 of C.botulinum. In another aspect said cell is Clostridium sporogenes,Clostridium perfringens, Clostridium acetobutylicum, B. cereus, B.thuringiensis, B. mycoidis, B. thermoproteolyticus, B. anthracis, B.megaterium, B. subtilis, E. coli, or a yeast cell. In one aspect, thepolypeptide of the present invention is modified inside the cell (i.e.glycosylated, phosphorylated, processed by proteases, etc.).Modification also includes the addition of non-proteinaceous co-factorsincluding metal-ions. Cells comprising the proteolytically inactivepolypeptide described above, any intermediate polypeptide product, aswell as the final proteolytically active polypeptide disclosed hereinare encompassed by this invention. Also encompassed by the presentinvention are cells comprising an inducer of expression of thepolypeptide of the present invention. Such an inducer of expression maybe a nucleic acid molecule or a polypeptide or a chemical entity,including a small chemical entity, having the effect of increasing theamount or activity of proteolytically active polypeptide of the presentinvention in cell cultures or lysates thereof. The inducer of expressionmay e.g. increase transcription or translation of a nucleic acidmolecule encoding the polypeptide of the present invention.Alternatively, the inducer of expression may be a compound capable ofactivating the proteolytically inactive polypeptide SEQ ID NO:2 or apolypeptide comprising a polypeptide sequence having at least 50%sequence identity with the sequence of SEQ ID NO: 2. In one aspect, saidcell comprises an inducer that is a proteolytically active polypeptidecapable of removing inhibitory amino acid residues from the N-terminusof said polypeptide. The inducer may be, for example, expressed byrecombinant means known to the person skilled in the art. Alternatively,the inducer may be isolated from a cell, e.g. a clostridial cell.

The present invention also relates to the use of the nucleic acidmolecule of the present invention for the manufacture of theproteolytically active polypeptide of the present invention.

In a related aspect, the present invention relates to a method for themanufacture of a proteolytically active polypeptide, comprising thesteps of: (a) chemically synthesising or translating from a nucleotidesequence a polypeptide, comprising a polypeptide sequence having atleast 50% sequence identity with the sequence of SEQ ID NO: 1; and (b)purifying the polypeptide of step (a.).

The term “chemically synthesising” means synthesising polypeptides bychemical means. Such methods are reviewed for example in Nilsson et al.,Ann. Rev. Biophys. Biomol. Struct. 2005. 34:91-118. The term “purifyingthe polypeptide” means removing from a mixture comprising thepolypeptide of the present invention compounds other than saidpolypeptide. The term also means removing the polypeptide of the presentinvention from a mixture comprising compounds other than saidpolypeptide. In a particular aspect, the term means separating theproteolytically active polypeptide from its proteolytically inactiveprecursor.

The nucleic acid may be translated in a cell or in a cell free system.Various systems for cell free translation are available to the skilledperson. The present invention encompasses, for example, the translationin a cell-free protein translation system comprising rabbit reticulocytelysate, wheat germ lysate, E. coli lysate, or other cellular lysates,for example lysates generated from C. botulinum and the like. Alsoencompassed is translating the polypeptide of the present invention fromthe nucleotide sequence of the present invention or the vector of thepresent invention. Transcription may be regulated or controlled by oneor more heterologous regulatory elements or by homologous regulatoryelements. Also encompassed by this aspect of the present invention isthe translation in a wildtype cell, i.e. a cell isolated from nature,such as any known isolate of C. botulinum, C. butyricum, C. baratii, andC. tetani. In a particular aspect, said cell is C. botulinum Hall strain(ATCC 3502). Various standard means and methods are available to theskilled person for bringing a nucleic acid molecule or a vector into thecell and for expressing the polypeptide of the present invention asrecombinant protein in a cell. Moreover, the skilled person knows manystandard techniques for isolating polypeptides from cells or celllysates or from cell free expression systems (e.g. Recombinant DNAPrinciples and Methodologies, J. Green, Marcel Dekker Inc., 1998; TheCondensed Protocols: From Molecular Cloning: A Laboratory Manual,Sambrook et al, Cold Spring Harbor Laboratory, 2006; Molecular Cloning:A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory,2000). Any of these means and methods may be used in the methods of thepresent invention.

The first polypeptide of the present invention may be translated from anucleic acid molecule encoding the proteolytically active polypeptide.SEQ ID NO: 26 is an example of such a nucleic acid molecule.Alternatively, said nucleic acid molecule may encode a precursorpolypeptide which is proteolytically inactive but which can be convertedinto the proteolytically active polypeptide of the present invention.SEQ ID NO: 27 is an example of such a nucleic acid molecule. Theproteolytically inactive precursor is also designated “inactiveBoNTHydrolase”, abbreviated iBH. This proteolytically inactivepolypeptide may e.g. be activated during or after translation or bycontacting, for example, said proteolytically inactive polypeptide witha protease capable of removing inactivating amino acid residues at theN-terminus of the proteolytically inactive polypeptide. An example of aproteolytically inactive polypeptide is the polypeptide represented bySEQ ID NO: 2. Another example is a polypeptide comprising a polypeptidesequence having at least 50% sequence identity with the sequence of SEQID NO: 2. The term “inactivating amino acid residues at the N-terminus”in one aspect relates to the first 248aa residues of said polypeptide.In another aspect, this term refers to a fragment of up to 10aa, 50aa,100aa, 150aa, 200aa, 250aa residues of said polypeptide. Any of thesepolypeptides are useful in the present invention's method for themanufacture of a proteolytically active polypeptide. In one aspect, theprotease capable of removing inactivating amino acid residues from theN-terminus of this polypeptide is isolated e.g. from Clostridiumbotulinum, Clostridium butyricum, Clostridium baratii, and Clostridiumtetani. In another aspect, the protease capable of removing saidinactivating amino acid is provided by providing a fractionated ornon-fractionated lysate of said cells. Inactivating amino acid residuesmay be removed by contacting the proteolytically inactive polypeptidewith said lysate and incubating until the proteolytically inactivepolypeptide is transformed into the proteolytically active polypeptide.

In another aspect of the present invention's method, the polypeptide istranslated in a cell. The cell may be a prokaryotic or eukaryotic cell.In one aspect the cell is selected from E. coli, B. subtilis or yeast.Also encompassed by the present invention is the translation of thepolypeptide of the present invention in a wildtype cell, i.e. a cellisolated from nature, such as any known isolate of Clostridiumbotulinum, Clostridium butyricum, Clostridium baratii, and Clostridiumtetani. In a particular aspect, said cell is C. botulinum Hall strain(ATCC 3502). In another particular aspect, said cell is the cell of thepresent invention described herein above.

Translation products obtained by the present invention's method may bepurified by various means, all of which are known to the person skilledin the art (e.g. Recombinant DNA Principles and Methodologies, J. Green,Marcel Dekker Inc., 1998; The Condensed Protocols: From MolecularCloning: A Laboratory Manual, Sambrook et al, Cold Spring HarborLaboratory, 2006; Molecular Cloning: A Laboratory Manual, Sambrook etal., Cold Spring Harbor Laboratory, 2000). Typical methods of purifyingthe polypeptide of the present invention may involve spinning of celllysate, ammonium sulphate precipitation of proteins, resuspension ofproteins, centrifugation of resuspended proteins, ion exchangechromatography, size exclusion chromatography, hydrophobic interactionchromatography and the like. Several combinations of such steps, indiffering order, may be useful for purifying the polypeptide of thepresent invention. A preferred method for purifying the polypeptide ofthe present invention is described in the Examples which illustrate theinvention.

In one aspect, the step of purifying comprises binding the polypeptideof the present invention to a solid support. The term “solid support”refers to a matrix encompassing e.g. silica, crosslinked dextran,crosslinked polyacrylamide or crosslinked agarose and the like. Alsoincluded are in particular polypeptides, glass, polystyrene,polypropylene, polyethylene, polyethylene glycol (PEG), dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. A solid support is, in an aspect of the invention, apolysaccharide matrix selected from the group consisting of: sepharose,sephadex, agarose, sephacell, micro-cellulose, and alginate-beads. Inanother aspect, said solid support can consist of glass-beads, and/orpolypeptide matrices.

In one aspect, the solid support is linked to the antibody of thepresent invention. The term “linked” means in one aspect stably linkedor stably associated. In another aspect, linked includes interactionssuch as indirect or direct, non-reversible or reversible, physical andchemical, electrostatic, and/or covalent bonds. In an aspect, theantibody is covalently linked, either directly or via a linker molecule,to the solid support. The antibody may be bound to said solid supportvia a linker, including small molecular compounds and peptide (orpolypeptide) linker molecules. The solid support can have virtually anypossible structural configuration or arrangement as long as the coupledantibody is capable of binding to its antigen. Thus, the matrix or solidsupport may be spherical, as in a bead, or cylindrical, as in the insidesurface of a test tube, or the external surface of a rod. Alternatively,the surface may be irregular or flat such as a sheet or test strip.

Said antibody linked to the solid support can be used e.g. in themanufacturing method of the present invention or in a diagnostic method.In one aspect, said manufacturing method may comprise a step of affinitychromatography, wherein said affinity chromatography is based on anantibody linked to a solid support. In one embodiment, said antibody isan antibody specifically binding to the proteolytically activepolypeptide of the present invention. In another embodiment, saidantibody is an antibody specifically binding to the proteolyticallyinactive polypeptide of the present invention.

In another aspect, the method for manufacturing the proteolyticallyactive polypeptide of the present invention comprises purifying thepolypeptide of the present invention from a mixture containingadditional components. Purification may be based on e.g. polarity,electrical charge and size. Therefore, the method may in one aspectcomprise one or more steps of separation selected from the groupconsisting of: normal-phase HPLC, reversed-phase HPLC,hydrophilic-interaction chromatography (HILIC), hydrophobic-interactionchromatography (HIC), ion-exchange chromatography (IEC) includinganion-exchange chromatography and cation-exchange chromatography,size-exclusion chromatography (SEC), gel-permeation chromatography(GPC).

In another aspect, said purification comprises the steps of: (a)separation by anion exchange chromatography; (b) separation by sizeexclusion chromatography; (c) separation by hydrophobic interactionchromatography; and (d) separation by size exclusion chromatography.

One or more fractions collected from a chromatography column can beconcentrated e.g. by precipitation or ultrafiltration.

In one aspect, the present invention relates to a composition comprisingthe proteolytically active polypeptide of the present invention. Usingthe method disclosed herein, it is possible to manufactureproteolytically active polypeptide of the present invention, which issubstantially free of proteolytically inactive polypeptide. In otherwords, the present invention's method provides a proteolytically activepolypeptide and a composition comprising no substantial contaminationwith inactive precursor protein of the polypeptide of the presentinvention. A composition is deemed to contain no substantialcontamination or to be substantially free of proteolytically inactiveprecursor polypeptide if, by using a western blot based detectionmethod, less than 5% of proteolytically inactive precursor can bedetected, wherein said 5% refer to the amount of proteolyticallyinactive precursor in relation to the sum of proteolytically active andinactive polypeptide. In another aspect, said composition issubstantially pure and comprises at least 50% proteolytically activepolypeptide of the present invention, wherein said 50% refers to theamount of proteolytically active precursor in relation to the totalamount of protein contained in the composition. In another aspect, saidsubstantially pure composition comprises at least 75%, 80%, 90% or atleast 98% proteolytically active polypeptide.

In another aspect, the present invention also relates to a polypeptidewhich is obtainable from the method for the manufacture of aproteolytically active polypeptide as described herein above and asillustrated in the Examples. Said proteolytically active polypeptide isin one aspect a proteolytically active polypeptide with the polypeptidesequence of SEQ ID NO: 1. In another aspect, the proteolytically activepolypeptide is a polypeptide having at least 50% sequence identity withthe sequence of SEQ ID NO: 1. In yet another aspect, the proteolyticallyactive polypeptide is a polypeptide which comprises a polypeptidesequence having at least 50% sequence identity with the sequence of SEQID NO: 1. The term “polypeptide which is obtainable”, as used hereinrefers in one aspect to a polypeptide that is translated from thenucleic acid of the present invention. The polypeptide can subsequentlyundergo posttranslational modification such as acylation, alkylation,amidation, amino acid addition, amino acid deletion, glycosylation,oxidation, S-glutathionylation, phosphorylation, sulphatation,proteolytic processing and the like. Moreover, the polypeptide may bindto a metal ion such as Li⁺, Na⁺, K⁺, Ag⁺, Cs⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺,Mn²⁺, Cu²⁺ or Zn²⁺. Preferably, said metal ion is Zn²⁺, Mn²⁺ or Co²⁺.

The present invention also relates in one aspect to an antibodyspecifically binding to the polypeptide of the present invention. Theterm “antibody” as used herein encompasses a monoclonal antibody, apolyclonal antibody, a single chain antibody, a human, humanized,primatized, or chimerized antibody, a bispecific antibody, a syntheticantibody, chemically or enzymatically modified derivatives, a fragmentof any of said antibodies or aptamers consisting of naturally occurringand/or chemically modified nucleic acids. Fragments of said antibodiesinclude F(ab′)2, F(ab), Fv or scFv fragments or chemically orenzymatically modified derivatives of any of these fragments.

In one aspect, the antibody of the present invention shall specificallybind to the proteolytically active polypeptide of the present inventionor its proteolytically inactive precursor. In one aspect, the antibodywhich is specific for the proteolytically active polypeptide of thepresent invention cross-reacts the proteolytically inactive polypeptidedescribed herein. In another aspect, the antibody is capable ofdiscriminating between the proteolytically active polypeptide of thepresent invention and its inactive precursor. In another aspect, theepitope for which said antibody is specific is located in an amino acidregion that is present in the proteolytically inactive polypeptide butnot in the proteolytically active polypeptide. For example, said epitopecan be an epitope of a polypeptide region consisting of amino acidresidues 1 to 248 of a polypeptide comprising the polypeptide sequencehaving at least 50% sequence identity with the sequence of SEQ ID NO: 2.

In another aspect, the epitope is formed by amino acid residues locatedN-terminal to amino acid 249 of a polypeptide comprising the polypeptidesequence having at least 50% sequence identity with the sequence of SEQID NO: 2. In another aspect, said epitope is removed from theproteolytically inactive polypeptide described herein by proteolyticprocessing.

In another aspect, the epitope for which the antibody of the presentinvention is specific is an epitope located at the N-terminus of apolypeptide comprising the polypeptide sequence having at least 50%sequence identity with the sequence of SEQ ID NO: 1. The term“N-terminus”, as used in this aspect of the invention, refers to aregion of the polypeptide comprising the N-terminal 50 amino acidresidues of said polypeptide sequence, preferably the N-terminal 25amino acid residues of said polypeptide sequence. In a particularaspect, the term refers to the N-terminal 14 amino acid residues. Theterm “epitope” as used herein relates to the antigenic determinant whichis recognised by the antibody of the present invention. In one aspect,the epitope is a linear epitope, in another aspect the epitope is aconformational epitope. In a particular aspect, the antigenicdeterminant consists of a peptide having the amino acid sequence of theN-terminus of the proteolytically active polypeptide of the presentinvention, wherein said peptide can have an amino acid length of 7 to14, preferably of 8, 9, 10, 11, 12, 13 or 14 amino acid residues.

The term “specifically binds” or “specifically binding to” in one aspectmeans that the antibody of the present invention does not cross-react toa significant extent with other epitopes either on the polypeptide ofthe present invention or on other polypeptides in general. Epitopespecificity is an important characteristic of the antibody of thepresent invention. Specificity of the antibody with respect to theproteolytically active versus proteolytically inactive polypeptide shallbe, in an aspect, at least 95%, at least 96%, at least 97%, at least98%, at least 99%. Specific binding can be tested by various well knowntechniques including, e.g., competition studies. Another importantcharacteristic is the sensitivity of the antibody. Sensitivity shall be,in one aspect of the invention, such that at least 70%, at least 80%, atleast 90%, at least 95% of the epitope comprised by a sample is bound.Sensitivity can be tested by well known techniques. Those skilled in theart will be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation. Conventionaltechniques for binding studies include radioimmunoassay, ELISA,equilibrium dialysis, isothermal microcalorimetry, BIACORE® assays(surface plasmon resonance, SPR) or other surface adsorption methods.The BIACORE® SPR system measures the antibody-antigen interaction. SPRresponse reflects a change in mass concentration at the detector surfaceas analytes bind or dissociate. Based on SPR, real-time BIACORE®measurements monitor interactions directly as they occur, seeBIAapplications Handbook, version AB (reprinted 1998), BIACORE® code No:BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998),BIACORE® code No: BR-1001-84. The binding properties such as sensitivityof an antibody of the present invention may, in principle, be determinedby binding studies using an immobilised antigen (the ligand) presentedon a sensor surface. The antibody to be tested (the analyte) will beprovided in the mobile phase, i.e. in a solution. In some cases, theantigen is attached indirectly to the surface through binding to anotherimmobilised molecule which is referred as the capturing molecule. Whenthe antibody is injected in a discrete pulse across the surface with theimmobilised antigens, essentially three phases can be subdivided: (i)Association of antibody with the antigen during sample injection; (ii)Equilibrium or steady state during sample injection, where the rate ofantibody binding is balanced by dissociation from the antibody-antigencomplex; (iii) Dissociation of antibody from the surface during bufferflow. It will be understood that such an assay can alternatively beperformed with immobilised antibodies to be investigated and an antigencontaining solution as the mobile phase. The association anddissociation phases provide information on the kinetics ofanalyte-ligand interaction (k_(a) and k_(d), the rates of complexformation and dissociation, k_(d)/k_(a)=K_(D)). The equilibrium phaseprovides information on the affinity of the analyte-ligand interaction(K_(D)). In an aspect of the invention, the antibody of the presentinvention has a K_(D) of less than 0.5 μM, in another aspect less than0.05 μM and, in another aspect, less than 0.02 μM.

The antibody as referred to in the present invention can be manufacturedby using methods which are described, e.g., in Harlow and Lane, 1988(Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988). Monoclonal antibodies can be prepared by thetechniques originally described in Kohler & Milstein, 1975 (Kohler &Milstein 1975, Nature 256: 495) and Galfre & Milstein, 1981 (Galfre &Milstein 1981, Meth Enzymol 73: 3). Said techniques comprise the fusionof mouse myeloma cells to spleen cells derived from immunized mammals.Antibodies can be further improved by techniques well known in the art.For example, surface plasmon resonance as employed in the BIACORE®system can be used to increase the efficiency of phage antibodies whichbind to the aforementioned epitope within polypeptide of the presentinvention (cf. Schier et al., 1996, Human Antibodies Hybridomas 7: 97;Malmborg et al., 1995, J. Immunol Methods 183: 7).

In an aspect of the invention, the antibody is produced by using apeptide comprising or consisting of the aforementioned epitope. Thepeptide can be produced e.g. synthetically or by recombinant expression.Alternatively, the antibody of the invention can be produced by applyingnatural occurring proteolytically active or inactive polypeptide of thepresent invention. In the latter case, it is to be understood that theresulting antibodies shall be further tested for specificity withrespect to the polypeptide of the present invention. In a further aspectof the invention, a monoclonal antibody of the invention is produced byusing a polypeptide of the present invention which can be treated by adetergent in order to make the epitope immunologically available.However, it will be understood that in a case were the antibody shall bedirected against a conformational epitope, no such detergent treatmentshall be carried out. In a further aspect, immune-stimulation agentssuch as keyhole limpet hemocyanin (KLH) may be also applied in suchprocess, especially when using a synthetic peptide.

The antibody of the present invention can be used, for example, foraffinity chromatography, immunoprecipitation, and immunolocalization ofthe polypeptide of the present invention as well as for the monitoringof the presence of said polypeptide in samples or in recombinantorganisms. Moreover, the antibody of the present invention may be usedin a detection method or in a diagnostic method. In a particular aspect,the antibody of the present invention is used in Western Blot or ELISA.In addition, the antibody of the present invention can be used intherapeutic applications. In particular, the antibody can be used forinhibiting the activity of the proteolytically active polypeptide of thepresent invention. Therefore, the antibody of the present invention alsohas various therapeutic applications described herein below.

The present invention also relates to the use of the proteolyticallyactive polypeptide of the present invention in a method forproteolytically processing a polypeptide. In one aspect, the presentinvention relates to a method for the manufacture of a proteolyticallyprocessed polypeptide, comprising the step of contacting: (a) a firstpolypeptide, said first polypeptide being the polypeptide of the presentinvention, with (b) a second polypeptide, said second polypeptide beingsusceptible to proteolysis by said first polypeptide, wherein saidcontacting results in proteolytic processing of said second polypeptideinto at least two cleavage products.

The present invention also relates to the use of Lys-N and/or Lys-Cand/or arginyl endopeptidase (endoproteinase Arg-C, LeR) from Lysobacterenzymogenes (ATCC 29487) (Wright D S, Graham L D, Jennings P A. BiochimBiophys Acta. 1998 Dec. 22; 1443(3):369-74). Moreover, also encompassedis the use of plasmin and/or omptin (OmpT), a membrane-bound serineprotease that cleaves at (Arg/Lys)-(Arg/Lys) motifs (K. Sugimura and T.Nishihara. J. Bacteriol. 170 (1988), pp. 5625-5632) in a method forproteolytically processing CNT such as BoNT/A. In one aspect, thepresent invention relates to a method for the manufacture of aproteolytically processed polypeptide, comprising the step ofcontacting: (a) a first polypeptide, said first polypeptide being Lys-Cor Lys-N, with (b) a second polypeptide, said second polypeptide beingsusceptible to proteolysis by said first polypeptide, wherein saidcontacting results in proteolytic processing of said second polypeptideinto at least two cleavage products, and wherein the second polypeptideis the single chain of BoNT/A. The term “Lys-C” refers to the 33 kDaserine endoproteinase Lys-C from Lysobacter enzymogenes (Lysylendopeptidase, LeK, Genbank acc. Q7M135) that specifically cleavespeptide bonds C-terminally to lysine or a homolog thereof having atleast 60% sequence identity. The term “Lys-N” refers to themetalloendopeptidase Lys-N isolated from Grifola frondosa and Pleurotusostreatus (Nonaka T et al., 1997, J Biol Chem. 272:30032-30039; Nonaka Tet al., 1998, J Biochem. 1998 124:157-162; Hori T et al., 2001, ActaCrystallogr D Biol Crystallogr. 57:361-368). Also encompassed by theterm are homologs of said protease having at least 60% sequenceidentity.

This method can be used, for example, for manufacturing proteolyticallyprocessed neurotoxin (CNT) or botulinum neurotoxin (BoNT). The term“BoNT”, as used throughout this invention, means botulinum neurotoxinand refers to neurotoxin obtainable from C. botulinum such as BoNT ofserotype A, B, C1, D, E, F or G. Also encompassed by the term “CNT” and“BoNT” is recombinant and modified neurotoxin comprising one or moremodifications including chemical modification or genetic modification.The term “genetic modification” means deletion, substitution or additionof one or more amino acid residues. Using the method of the presentinvention, it is now possible to obtain neurotoxin compositions withsignificantly less contamination by unprocessed or partially processedneurotoxin, since those contaminants are efficiently processed intodi-chain neurotoxin. In one aspect, the di-chain neurotoxin is a nativedi-chain neurotoxin, wherein the C-terminus of the light chain and theN-terminus of the heavy chain are identical to the corresponding fullyprocessed di-chain neurotoxin isolated from wildtype clostridia.

The term “contacting” as used herein refers to bringing at least twodifferent compounds in physical proximity as to allow physical and/orchemical interaction of said compounds. In accordance with the method ofthis invention, the said two different compounds are, in an aspect, thefirst and the second polypeptide which are comprised by the solution.Contacting is carried out under conditions and for a time beingsufficient to allow interaction of the first and second polypeptide. Theterm “proteolytically processed polypeptide” as used herein refers inone aspect to a polypeptide, the polypeptide chain of which has beenhydrolysed or cleaved at one or more peptide bonds. In another aspect,the term refers to a polypeptide that has been proteolytically cleavedby an endoproteinase or endopeptidase. In another aspect, the termrefers to a polypeptide which has been cleaved to a degree of at least50%. In another aspect, said proteolytically processed polypeptide isthe second polypeptide. In another aspect, at least 60%, 70%, 80%, 90%or 95% are proteolytically processed.

The term “first polypeptide”, as used herein, refers to the polypeptideof the present invention, i.e. the proteolytically active or activatedpolypeptide, also designated “active BoNTHydrolase”. Since the activeBoNTHydrolase can be obtained from the supernatant of C. botulinum, itwas initially termed native BoNTHydrolase, abbreviated “nBH”. However,the term “first polypeptide” and “nBH” also refers to BoNTHydrolasesthat are obtainable from other sources. The term “second polypeptide”,as used herein, refers to the substrate of said first polypeptide. Theterm “being susceptible to proteolysis” refers to a feature orrequirement of the second polypeptide and is used herein meaning thatthe second polypeptide is proteolytically cleavable by said firstpolypeptide. In other words, the term “being susceptible to proteolysis”means that the second polypeptide comprises a protease recognition andcleavage site allowing it to function as a substrate of the firstpolypeptide. The “second polypeptide” is a substrate of the firstpolypeptide and is proteolytically processed into two or more cleavageproducts. Using the assay described herein above, the skilled person cantest whether a given polypeptide is a substrate of the first polypeptideand, thus, a “second polypeptide” according to present invention'sdefinition. The term “at least two cleavage products” includes, forexample, up to two, three, four, five and up to six cleavage products.

This method can be used, for example, for preparing a pharmaceuticalcomposition comprising clostridial neurotoxin or for generatingpolypeptide fragments used in a method of mass spectrometry. The firstpolypeptide and the second polypeptide can be contacted at various stepsin the manufacturing process of proteolytically processed polypeptide.In one aspect, the step of contacting the first polypeptide and thesecond polypeptide is within a cell. In a particular aspect of thisembodiment, the first and the second polypeptide are expressed in saidcell.

In another aspect, said step of contacting is in a cell lysate or in apurified cell lysate. This aspect encompasses adding the firstpolypeptide to the lysate or the purified lysate. The first polypeptidecan be added at various steps during purification of the secondpolypeptide from the cell lysate. For example, the first polypeptide canbe added prior to or after: protein precipitation, ion exchangechromatography, hydrophobic interaction chromatography and/or sizeexclusion chromatography. Moreover, also encompassed is the addition ofthe first polypeptide to a pharmaceutical composition. In this aspect,the polypeptide of the present invention is used e.g. forproteolytically cleaving the second polypeptide e.g. for activating asecond polypeptide which is a therapeutic agent contained in thepharmaceutical composition. Also envisaged is the administration of thefirst polypeptide to a subject, in order to proteolytically process asecond polypeptide in the subject. Administration also includesco-administration of the first and the second polypeptide. Alsoencompassed by this method is a step of incubation at conditions and fora time sufficient to cleave the second polypeptide. In one aspect, theconditions can comprise adding a buffer selected from the groupconsisting of 100 mM Tris-HCl, pH 8.0 or PBS (50 mM Na₂HPO₄, 150 mMNaCl, pH 7.4). Preferred buffer conditions include 100 mM Tris-HCl, pH8.0. The “time sufficient to cleave” can be determined using the assaydescribed herein above. In one aspect, said “time sufficient to cleave”depends on the degree of cleavage that the proteolytically processedpolypeptide or a composition comprising it should have. In one aspect,the method comprises a step of incubating the first and the secondpolypeptide for at least 30 min, 60 min, 120 min or at least 240 min. Inanother aspect, the first and second polypeptide are incubated for up to30 min, 60 min, 120 min, 240 min, 480 min or up to 600 min. In anotheraspect, the method comprises a step of incubating the first and thesecond polypeptide at 4° C. or at 37° C. In another aspect, the methodcomprises a step of incubating for up to 1 h, up to 2 h, 4 h, 6 h, 10 hor up to 16 h.

In one aspect, the polypeptide chain of said second polypeptidecomprises a sequence selected from any one of SEQ ID NOs: 4 to 25. In amore particular aspect, the polypeptide chain of said second polypeptidecomprises a sequence selected from any one of SEQ ID NOs: 4 to 25 andwherein the second polypeptide is cleaved C-terminal to a basic aminoacid residue within said sequence of any one of SEQ ID NOs: 4 to 25.Said sequences represent amino acid sequences of known substrates of theproteolytically active polypeptide of the present invention. As shownherein, said substrates are cleaved C-terminal to a basic amino acidresidue contained in the sequence, compare Table 1, column LC and H_(N).In a preferred aspect, said second polypeptide comprises a sequenceselected from SEQ ID NO: 4 to 10. In another preferred aspect, saidsecond polypeptide is BoNT/A or a derivative thereof, including e.g. thepolypeptide of SEQ ID NO: 3 and derivatives thereof. The term“derivative” as used with respect to this and other aspects of theinvention, comprises amino acid mutations such as addition,substitution, deletion or truncation of one or more amino acid residues.

In one aspect, the second polypeptide comprises a derivative of any oneof SEQ ID NOs: 4 to 25, or of SEQ ID NO: 3, wherein said derivative hasone or more point mutation and/or one or more additional amino acidresidues. In another aspect, said derivative has up to 1, up to 2, up to3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to15 point mutations. By using the activity assay for determining proteaseactivity, as described herein, the skilled person can determine whethera given derivative is processed by the proteolytically activepolypeptide of the present invention. In another aspect, the derivativecontains a point mutation changing a basic amino acid residue into anon-basic amino acid residue. In another aspect, the derivative has atleast 50% sequence identity with any one of SEQ ID NOs: 4 to 25. Inanother aspect, said derivative or a polypeptide comprising thederivative is a substrate of the first polypeptide and isproteolytically cleavable by the first polypeptide. A typical example isa derivative of SEQ ID NO: 3 comprising e.g. one or more point mutationsin the light or heavy chain.

In another aspect, said second polypeptide comprises (a) a polypeptidesequence having at least 30% sequence identity with the sequence of SEQID NO: 3 [(BoNT/A of ATCC 3502, Genbank acc. AAA23262)]; or (b) apolypeptide sequence selected from the group consisting of Tetanusneurotoxin, protein of the coagulation cascade Factor X or Prothrombin(Factor II), digestive enzymes of the pancreas like trypsin,chymotrypsin, pepsin, papain. At least 30% means at least 30%, at least40%, at least 50%, at least 85%. In a particular aspect, the sequenceidentity of said second polypeptide sequence having at least 50%sequence identity with the sequence of SEQ ID NO: 3 is determined basedon amino acid position 420 to 466 of SEQ ID NO: 3, in another aspect,said sequence identity is determined based on any one of SEQ ID NOs: 4to 25. In other words, said aspect refers to a second polypeptide whichcomprises a polypeptide sequence which has e.g. at least 30% sequenceidentity to the polypeptide sequence found between amino acid positions420 to 466 of SEQ ID NO: 3 or at least 30% sequence identity to thepolypeptide sequence of any one of SEQ ID NOs: 4 to 25. A polypeptideaccording to this definition is, e.g. obtainable from C. botulinum, C.tetani or C. sporogenes. Said second polypeptide may be, for example, anaturally occurring neurotoxin such as BoNT/A, B, C1, D, E, F or G or aderivative thereof comprising one or more amino acid mutations such asaddition, substitution, deletion or truncation of one or more amino acidresidues. Encompassed are e.g. derivatives lacking e.g. the nativeneurotoxin H_(C) domain or parts thereof or derivatives with other aminoacid residues replacing the neurotoxin H_(C) domain as well asderivatives with an additional light chain or another proteinaceouscargo molecule fused N-terminally to the light chain of BoNT.

In another aspect, the second polypeptide may contain additional aminoacid residues at the N- or C-terminus or at an internal position. Theadditional amino acid residues may be flanked by one or more proteasecleavage sites. In another aspect, the additional amino acid sequencefunctions as a detectable tag and/or allows binding to a solid support.An example is a his tag or a GST tag. Another example is the amino acidsequence VPPTPGSAWSHPQFEK containing the Streptag, preferably added tothe C-terminus.

In a particular aspect, said second polypeptide is a polypeptidecomprising a polypeptide sequence as shown in GenBank no: CBZ04958.1,YP_002805603.1, ZP_02994746.1, YP_001788403.1, YP_001782718.1,ZP_02616437.1, ZP_02614241.1, YP_001392361.1, YP_001255575.1 or ahomolog thereof having at least 50% sequence identity.

In another aspect, the biological activity of said second polypeptide ismodulated by the proteolytic cleavage. It is well known to the skilledperson, that the function of many polypeptides can be modulated byproteolytic processing. “Modulated” as used herein means increased ordecreased, activated or inactivated. For example, the biologicalactivity of many clostridial neurotoxins is increased or triggered byproteolytically processing a single chain neurotoxin into a di-chainneurotoxin, wherein the di-chain neurotoxin is composed of a light and aheavy polypeptide chain, which are covalently linked through adisulfide-bridge. The biological activity of the neurotoxin encompassesat least three separate activities: the first activity is a “proteolyticactivity” residing in the light chain of the neurotoxin and isresponsible for hydrolysing the peptide bond of one or more polypeptidesinvolved in the regulation of cellular membrane fusion. A secondactivity is a “translocation activity”, residing at the N-terminal endof the heavy chain of the processed neurotoxin and is involved in thetransport of the light chain across the lysosomal membrane and into thecytoplasm. A third activity is a “receptor binding activity”, residingat the C-terminal end of the heavy chain of the processed neurotoxin andinvolved in binding and uptake of the neurotoxin to a target cell. In apreferred aspect, the term biological activity as used herein meansproteolytic activity. In a more preferred aspect, the term meansincreased proteolytic activity.

Biological activity of clostridial neurotoxin can be measured by varioustests, all of which are known to the person skilled in the art. Thesetests allow determining one or more of the activities mentioned above.For example, the mouse LD₅₀ assay or the ex vivo mouse phrenic nervehemidiaphragm (MPN) assay as described by Pearce et al., 1994 (Pearce LB, Borodic G E, First E R, MacCallum R D (1994), Toxicol Appl Pharmacol128: 69-77) and Habermann et al., 1980 (Habermann E, Dreyer F, BigalkeH. (1980), Naunyn Schmiedebergs Arch Pharmacol. 311:33-40) allowdetermining the toxic effect of a given neurotoxin preparation on aliving organism or an isolated neuromuscular preparation. Forestablishing the toxic effect in an LD₅₀ assay, the neurotoxin must bebiologically active in each of said three activities mentioned above.Moreover, various other assays are available, allowing e.g. to determinewhether a neurotoxin or the light chain of the neurotoxin isproteolytically active. Such assays are e.g. based on contacting BoNT/Awith SNAP-25. Alternatively, a peptide representing the cleavage site ofSNAP-25 can be used, wherein the peptide can be labelled to easedetection. In a preferred aspect, biological activity is determined byusing the MPN assay described herein above.

In another aspect, said first polypeptide is activated byproteolytically processing an inactive precursor polypeptide, saidinactive precursor polypeptide comprising a polypeptide sequence havingat least 60% sequence identity with the sequence of SEQ ID NO: 2. Thisaspect rests on the observation that a polypeptide having thepolypeptide sequence of SEQ ID NO: 2 is proteolytically inactive, whileN-terminal truncations thereof are proteolytically active. It is alsoenvisaged by the present invention to use the proteolytically inactivepolypeptide in the methods described herein. The proteolyticallyinactive polypeptide described herein can be activated e.g. by removinga fragment from the N-terminus or the entire N-terminus comprising aminoacid residues 1 to 248 of SEQ ID NO: 2. In one aspect, the N-terminus isremoved by a protease, in another aspect, the N-terminus is removed byautoproteolysis of SEQ ID NO: 2. 60% sequence identity refers to asequence alignment with full length NT02CB1447.

In another aspect, the present invention's method for the manufacture ofa proteolytically processed polypeptide comprises the step of purifyingthe proteolytically processed second polypeptide or at least one or twoor more cleavage products thereof. Purification of C. botulinumexpressed BoNT/A may be done e.g. as essentially described in the priorart (DasGupta 1984, Toxicon 22, 415; Sathyamoorthy 1985, J BiolChemistry 260, 10461). In particular, purification of the neurotoxin cancontain one or more precipitation- and extraction steps, one or moreconcentration steps, and further distinct chromatographic steps.Recombinant single chain BoNT/A and its purification is described inprior art (Rummel et al., 2004, Mol Microbiol. 51:631-43).

In a preferred embodiment, the Clostridium strain is C. botulinum, forexample producing BoNT/A, or a derivative thereof. For fermentation, theprocess described by DasGupta B. R. et al. in Toxicon, vol. 22, No. 3,p. 414 to 424, 1984, can be used. Therefore 0.5% yeast extract and 0.6%autoclaved yeast paste is added to 2% of the N—Z-amine type A medium,and a pH of 7.2 will be adjusted with the help of 4N NaOH, and themedium prepared in such a way will afterwards be autoclaved. To thismedium separately autoclaved glucose (20% by weight per volume) may beadded, to come to a final concentration of glucose of 0.5% in themedium. Incubation may occur e.g. at 37° C. without stirring, whereinthe fermentation is discontinued e.g. after 96 hours. It is within thescope of the present invention that besides the batch fermentationdescribed before also semi-batch fermentation, repeated batchfermentation or continuous fermentation can be performed.

After the actual fermentation and separation of the fermentation mediumfrom the cells the fermentation medium may undergo a first precipitationwith the goal of removing large proteins. The precipitation ispreferably an acid precipitation. Reaction conditions for such an acidprecipitation are known to those skilled in the art. Typically 1.5 MH₂SO₄ may be used, to acidify the supernatant to a pH of 3.5. Thecentrifugation usually occurs for 20 minutes at 2400×g at 4° C. Thepellet received through centrifugation may be washed with water,preferably repeatedly. Subsequently, the pellet may be extracted with a0.1 M citric acid-trisodium citrate buffer, pH 5.5 e.g. for an hour.Subsequently, a further centrifugation step may be performed, e.g. at9800×g for 20 minutes at 4° C. The so obtained pellet optionally canthen again be extracted as described before. The supernatant of theextraction, and both supernatants in case of repetition of theextraction, may then be subjected to protamine sulphate precipitation.The precipitation may continue overnight, e.g. at 8° C. Subsequently,the precipitate may be centrifuged, e.g. for 20 minutes at 4° C. and at12,000×g. The supernatant of centrifugation may be subject to aprecipitation such as an ammonium sulphate precipitation, whereby otherlarger proteins can be removed. After the ammonium sulphateprecipitation step another centrifugation step may be added andsubsequently the so obtained pellet may be redissolved and, optionally,be subjected to a dialysis. The extract which is preferably dialysed andcentrifuged again, can be subjected to a succession of chromatographysteps with the objective of purifying the neurotoxin. Each of thechromatography steps serves to remove contaminants such as protaminesulphate, remaining DNA, parts of smaller proteins and middle-sizedproteins as well as the hemagglutinins of the botulinum neurotoxinprotein complex. For this purpose, one or more chromatography steps maybe used in a preferred embodiment. Optionally, the eluate of, e.g. thelast chromatography step, may be filtrated in order to reduce germs.Optionally the eluate can be diluted before filtration and suitableadjuvants can be added. During further steps another sterile filtrationmay be carried out after addition of the adjuvants. In one aspect, thefiltration is carried out in reaction containers which may then besubject to a step of lyophilization.

The present invention also relates to a composition obtainable by thepresent invention's method for the manufacture of a proteolyticallyprocessed polypeptide. In one aspect, said composition comprises amixture of processed and unprocessed second polypeptide, wherein saidmixture may contain less than 5%, 4%, 3%, 2% or less than 1% unprocessedsecond polypeptide. In an aspect of said composition, said secondpolypeptide is BoNT or a derivative thereof. The BoNT can e.g. beselected from group consisting of BoNT of serotype A, B, C, D, E, F andG, including a derivative thereof. The composition can be e.g. a liquidor a solid composition and may contain one or more carrier, adjuvantsand/or excipients.

In another aspect, the present invention also relates to a method forthe manufacture of a medicament, i.e. a pharmaceutical composition,comprising the steps of the aforementioned method and the further stepof formulating the purified di-chain neurotoxin as medicament. In oneaspect, said medicament comprises a mixture of processed and unprocessedsecond polypeptide, wherein said mixture contains less than 5%unprocessed second polypeptide. In preferred embodiments, the mixturecontains less than 4%, 3%, 2% or less than 1% unprocessed secondpolypeptide.

The present invention also relates to various medical uses of thecompounds disclosed herein:

In one aspect, the present invention relates to a proteolytically activepolypeptide according to the present invention for use as a medicamentor in a pharmaceutical composition.

In another aspect, the present invention relates to a compositionaccording to the present invention for use as a medicament or inpharmaceutical composition.

In yet another aspect, the present invention relates to an antibodyaccording to the present invention for use as a medicament or inpharmaceutical composition.

In yet another aspect, the present invention relates to an inhibitoraccording to the present invention for use as a medicament or inpharmaceutical composition.

In particular, the present invention relates to a pharmaceuticalcomposition comprising the polypeptide of the present invention, theantibody of the present invention, the composition of the presentinvention or the inhibitor of the present invention.

The term “composition” as used herein refers to any compositionformulated in solid, liquid, aerosol (or gaseous) form and the like.Said composition comprises e.g. a therapeutically active compound of theinvention optionally together with suitable auxiliary compounds such asdiluents or carriers or further ingredients. In one aspect, thetherapeutically active compound is the proteolytically activepolypeptide of the present invention. In another aspect, the therapeuticcompound is the proteolytically processed second polypeptide asdescribed herein above such as the di-chain neurotoxin. In anotheraspect, the therapeutically active compound is the antibody of thepresent invention. In another aspect, the therapeutically activecompound is the inhibitor of the proteolytically active polypeptide ofthe present invention.

In this context, it is distinguished for the present invention betweenauxiliary compounds, i.e. compounds which do not contribute to theeffects elicited by the compound of the present invention uponapplication of the composition for its desired purpose, and furtheringredients, i.e. compounds which contribute a further effect ormodulate the effect of the compound of the present invention. Suitablediluents and/or carriers depend on the purpose for which the compositionis to be used and the other ingredients. The person skilled in the artcan determine such suitable diluents and/or carriers without furtherado.

The carrier(s) must be acceptable in the sense of being compatible withthe other ingredients of the formulation and being not deleterious tothe recipient thereof. The pharmaceutical carrier employed may include asolid, a gel, or a liquid. Exemplary of solid carriers are lactose,terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesiumstearate, stearic acid and the like. Exemplary of liquid carriers arephosphate buffered saline solution, syrup, oil, water, emulsions,various types of wetting agents, and the like. Similarly, the carrier ordiluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax. Saidsuitable carriers comprise those mentioned above and others well knownin the art, see, e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa.

The diluent(s) is/are selected so as not to affect the biologicalactivity of the combination. Examples of such diluents are distilledwater, physiological saline, Ringer's solutions, dextrose solution, andHank's solution, in addition, the pharmaceutical composition orformulation may also include other carriers, adjuvants, or non-toxic,non-therapeutic, non-immunogenic stabilisers and the like.

In one aspect, a pharmaceutical composition as used herein comprises thebiologically active neurotoxin obtained by the method of the presentinvention, optionally, one or more pharmaceutically acceptable carrier.The active neurotoxin can be present in liquid or lyophilized form. Inan aspect, said compound can be present together with glycerol, proteinstabilisers (e.g., human serum albumin (HSA)) or non-proteinaceousstabilisers such as polyvinylpyrrolidone or hyaluronic acid. Thepharmaceutical composition is, in one aspect, administered topically.Conventionally used drug administration is administered intramuscular,subcutaneous (near glands). However, depending on the nature and themode of action of a compound the pharmaceutical composition may beadministered by other routes as well. The di-chain neurotoxinpolypeptide is the active ingredient of the composition, and is in oneaspect administered in conventional dosage forms prepared by combiningthe drug with standard pharmaceutical carriers according to conventionalprocedures. These procedures may involve mixing, granulating, andcompression, or dissolving the ingredients as appropriate to the desiredpreparation. It will be appreciated that the form and character of thepharmaceutical acceptable carrier or diluent is dictated by the amountof active ingredient with which it is to be combined, the route ofadministration, and other well-known variables.

A therapeutically effective dose refers to an amount of the compound,the neurotoxin, to be used in a pharmaceutical composition of thepresent invention which prevents, ameliorates or treats the symptomsaccompanying a disease or condition referred to in this specification.Therapeutic efficacy and toxicity of the compound can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED50 (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀.

The dosage regimen will be determined by the attending physician andother clinical factors. As is well known in the medical arts, dosagesfor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Progress can be monitoredby periodic assessment. The pharmaceutical compositions and formulationsreferred to herein are administered at least once in order to treat orameliorate or prevent a disease or condition recited in thisspecification. However, the said pharmaceutical compositions may beadministered more than one time.

In a further aspect of the invention, the aforementioned composition isa medicament or a cosmetic composition. In one aspect the saidmedicament comprising the biologically active neurotoxin can be used forprevention and/or treatment of at least one of the following diseasesand disorders: voluntary muscle strength, focal dystonia, includingcervical, cranial dystonia, and benign essential blepharospasm,hemifacial spasm, and focal spasticity, gastrointestinal disorders,hyperhidrosis, and cosmetic wrinkle correction, in a further aspect alsoblepharospasm, oromandibular dystonia, jaw opening type, jaw closingtype, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid,opening cervical dystonia, antecollis, retrocollis, laterocollis,torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodicdysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodicdyspnea, limb dystonia, arm dystonia, task specific dystonia, writer'scramp, musician's cramp, golfer's cramp, leg dystonia, thigh adduction,thigh abduction knee flexion, knee extension, ankle flexion, ankleextension, equinovarus, deformity foot dystonia, striatal toe, toeflexion, toe extension, axial dystonia, pisa syndrome, belly dancerdystonia, segmental dystonia, hemidystonia, generalised dystonia,dystonia in lubag, dystonia in corticobasal degeneration, dystonia inlubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia inParkinson's disease, dystonia in Huntington's disease, dystonia inHallervorden Spatz disease, dopa-induced dyskinesias/dopa-induceddystonia, tardive dyskinesias/tardive dystonia, paroxysmaldyskinesias/dystonias, kinesiogenic non-kinesiogenic action-inducedpalatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps,hereditary chin trembling, paradoxic jaw muscle activity,hemimasticatory spasms, hypertrophic branchial myopathy, maseterichypertrophy, tibialis anterior hypertrophy, nystagmus, oscillopsiasupranuclear gaze palsy, epilepsia, partialis continua, planning ofspasmodic torticollis operation, abductor vocal cord paralysis,recalcitant mutational dysphoria, upper oesophageal sphincterdysfunction, vocal fold granuloma, stuttering Gilles de la Tourettesyndrome, middle ear myoclonus, protective larynx closure,postlaryngectomy, speech failure, protective ptosis, entropion sphincterOdii dysfunction, pseudoachalasia, nonachalsia, oesophageal motordisorders, vaginismus, postoperative immobilisation tremor, bladderdysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm,hemifacial spasm, reinnervation dyskinesias, cosmetic use craw's feet,frowning facial asymmetries, mentalis dimples, stiff person syndrome,tetanus prostate hyperplasia, adipositas, treatment infantile cerebralpalsy strabismus, mixed paralytic concomitant, after retinal detachmentsurgery, after cataract surgery, in aphakia myositic strabismus,myopathic strabismus, dissociated vertical deviation, as an adjunct tostrabismus surgery, esotropia, exotropia, achalasia, anal fissures,exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome,hyperhidrosis, axillar palmar plantar rhinorrhea, relativehypersalivation in stroke, in Parkinson's, in amyotrophic lateralsclerosis spastic conditions, in encephalitis and myelitis autoimmuneprocesses, multiple sclerosis, transverse myelitis, Devic syndrome,viral infections, bacterial infections, parasitic infections, fungalinfections, in hereditary spastic paraparesis postapoplectic syndromehemispheric infarction, brainstem infarction, myelon infarction,migraine, in central nervous system trauma, hemispheric lesions,brainstem lesions, myelon lesion, in central nervous system hemorrhage,intracerebral hemorrhage, subarachnoidal hemorrhage, subduralhemorrhage, intraspinal hemorrhage, in neoplasias, hemispheric tumors,brainstem tumors, myelon tumors, snoring (WO 2000/033863). For detailsand symptoms see, e.g., Jost 2007, Drugs 67(5), 669 or Dressler 2000 inBotulinum Toxin Therapy, Thieme Verlag, Stuttgart, New York.

In another aspect of the invention, the composition is a cosmeticcomposition which can be formulated as described for a pharmaceuticalcomposition above. For a cosmetic composition, likewise, it is envisagedthat the compound of the present invention is in an aspect used insubstantially pure form. Cosmetic compositions are, in a further aspect,to be applied intramuscular. In an even further aspect of the invention,cosmetic compositions comprising the neurotoxin can be formulated as ananti-wrinkle solution.

In another aspect, the pharmaceutical composition comprises the antibodyor the inhibitor of the present invention. Since the present invention'spolypeptide is responsible for activating clostridial neurotoxins, theantibody will be useful for reducing the toxic effect observed duringinfection with clostridia. Therefore, the antibody of the presentinvention can in one aspect be used for treating an infection byClostridia, including Clostridium perfringens, Clostridium difficile,Clostridium tetani, Clostridium botulinum, Clostridium baratii,Clostridium butyricum, Clostridium sporogenes, Clostridiumacetobutylicum, Clostridium haemolyticum, Clostridium novyi andClostridium oedematiens. Furthermore, the antibody of the presentinvention can in another aspect be used for the treatment of symptomsassociated with said infection. Moreover, said antibody can be used inthe treatment of a condition or a symptom associated with the condition,wherein the condition is selected from Botulism, Tetanus,Pseudomembranous colitis, Gangrene, Food poisoning and the like.

In another aspect, the pharmaceutical composition comprises theproteolytically active polypeptide of the present invention. Saidpharmaceutical composition can be used in one aspect for proteolyticallycleaving polypeptides involved in coaglutination, in particular fortreating patients with hypocoaglutination. In another aspect, thepharmaceutical composition can be used as fibrinolyticum, in particularfor treating patients with myocard infarct, pulmonary embolism, deepvenous thromboembolism, i.e. for removing blood clots. Also envisaged isthe use of the pharmaceutical composition in the treatment of stroke.Moreover, in other aspects, the pharmaceutical composition can be usedin the treatment of exokrine pancreatic insufficiency for replacingeither of trypsin, chemotrypsin, pepsin. Moreover, in other aspects, thepharmaceutical composition can be used in the treatment of patientsaffected by inflammatory reactions, in the treatment of cancer patients,in particular for proteolytically cleaving surface exposed tumorantigens. Moreover, in another aspect, the pharmaceutical compositioncan be used in the treatment of papilloma.

The present invention also relates to a method of screening for aninhibitor comprising the step of (a) contacting the proteolyticallyactive polypeptide of the present invention with a known substrate and,optionally, with a putative inhibitor; and (b) determining the effect ofthe putative inhibitor on the conversion of substrate into cleavageproduct, wherein a reduction in the amount of cleavage product isindicative for the inhibitory effect of the putative inhibitor. In oneaspect the putative inhibitor is a peptide comprising an amino acidsequence selected from any one of SEQ ID NOs: 4 to 25, wherein at leastone basic amino acid of said amino acid sequence is replaced with anon-basic amino acid. In a further aspect, said peptide comprises one ormore chemical modifications. In another aspect, the inhibitor is apeptidomimetic of said peptide. In another aspect, the putativeinhibitor is part of a chemical compound microarray, i.e. a collectionof organic chemical compounds. In yet another aspect, the inhibitor isthe antibody of the present invention. This method is useful foridentifying compounds capable of inhibiting the proteolytically activepolypeptide of the present invention. An initial screen may be based on,for example, a peptide comprising an amino acid sequence selected fromany one of SEQ ID NOs: 4 to 25. Peptides capable of inhibiting thepolypeptide of the present invention may be modified in order toincrease inhibition. Modifications include amino acid substitution orchemical modifications. Typically, this method is carried out bycontacting the polypeptide of the present invention with a knownsubstrate in the presence and absence of a putative inhibitor (step (a)of the method) and by comparing the effect of the putative inhibitor onthe conversion of substrate into cleavage product. A reduction of theconversion rate in the presence of putative inhibitor is indicative ofan inhibitory effect.

The present invention also relates to an inhibitor of theproteolytically active polypeptide of the present invention, whereinsaid inhibitor is (a) an inhibitor comprising an amino acid sequence asshown in any one of SEQ ID NOs: 4 to 25, wherein a basic amino acidcontained therein is replaced with a non-basic amino acid; or (b) theantibody of the present invention.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

The figures show:

FIG. 1: Activity test of fractions collected from HiPrep 16/10 Q FF.

5 μl of fractions collected from HiPrep 16/10 Q FF run were analysed forenzymatic activity by incubating 2 μg scBoNTA (lane 2) for 1 h at 37° C.and subsequent 10% SDS-PAGE. Lane 1: low molecular weight marker (LMW):116 kDa, 66 kDa, 45 kDa, 35 kDa.

FIG. 2: Analysis of collected fractions from SEC (HiLoad 16/60 Superdex75) regarding content of nBH with a molecular weight of ˜37.3 kDa by12.5% SDS-PAGE.

Fractions 9 to 11 contain the majority of nBH. (Lane 1: LMW: 116 kDa, 66kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4 kDa)

FIG. 3: 12.5% SDS-PAGE analysis for determination of purity and proteinconcentration of three purification batches of nBH.

lane 1, LMW (116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4kDa); lane 2, nBH Lot TE311206 (192 ng/μl maturated NT02CB1446/CBO1444,amino acids 254-594 of Genbank acc. CAL82987.1, MW: 38.6 kDa); lane 3,nBH Lot TIK301009 (130 ng/μl maturated NT02CB1447/CBO1445, SEQ ID NO: 1,amino acids 249-581 of Genbank acc. CAL82988.1, MW: 37.3 kDa); lane 4,nBH Lot TIK280509 (114 ng/μl maturated NT02CB1447/CBO1445, SEQ ID NO: 1,amino acids 249-581 of Genbank acc. CAL82988.1, MW: 37.3 kDa).

FIG. 4: ESI-MS/MS spectrum analysis report.

38.6 kDa protein band of nBH lot TE311206 was identified asNT02CB1446/CBO1444 with a Mascot score of 725 and a peptide MS/MSsequence coverage of 29.6% over the entire open reading frame (ORF). Nopeptide (grey box, identified MS peptide; red squares, identified aminoacid y-/b-ion of peptide after MS/MS decay) was identified derived fromthe N-terminal 253 amino acids. The MS/MS analysis of lot TE311206displayed a sequence coverage of 52% according to the C-terminal aminoacids 254-594 forming the nBH.

FIG. 5: ESI-MS/MS spectrum analysis report.

37.3 kDa protein band of nBH lot TIK301009 was identified asNT02CB1447/CBO1445 with a Mascot score of 555 and a peptide MS/MSsequence coverage of 28.4% over the entire open reading frame (ORF).Except one all peptides (grey box, identified MS peptide; red squares,identified amino acid y-/b-ion of peptide after MS/MS decay) identifiedderive from the C-terminal 333 amino acids. The MS/MS analysis of lotTIK301009 displayed a sequence coverage of 49.5% according to theC-terminal amino acids 249-581 forming the nBH.

FIG. 6: Comparison of concentration dependent proteolytic activity ofnBH derived from three purification batches.

A 12.5% SDS-PAGE of activity test analysing nBH derived from the batchesTIK301009, TIK280509 and TE311206 using the following dilutions of theconcentrated nBH: 1:10, 1:30, 1:100, 1:300, 1:1000. The assay wasperformed by incubating 1 μg scBoNT/A and 2 μl dH₂O and 1 μl ofcorrespondingly diluted nBH for 60 min at 37° C. For SDS-PAGE analysis,3 μl of a reducing 4×SDS Laemmli buffer was added to a final volume of10 μl. 150 kDa scBoNT/A was cleaved into 100 kDa heavy chain and 50 kDalight chain.

B The optical density of the protein bands of heavy chain, light chainand scBoNT/A were quantified and the sum of light and heavy chainproduct bands was divided by sum of LC, HC and scBoNT/A protein bands.Higher dilution of first polypeptide decrease cleavage rate. Thespecific proteolytic activity of the three different batches is nearlyidentical

FIG. 7: Time dependent cleavage of scBoNT/A wild-type and mutantscontaining a modified loop by nBH.

A Modification of the loop sequence. In scBoNTAS Throm all lysineresidues are removed and the thrombin recognition sequence LVPRGS isinserted whereas in scBoNT Res the loop lacks any basic amino acids.Shortening the loop to eight small residues or five amino acids withbulky side chains yields scBoNTAS (GGSG)₂ and scBoNTAS FQWYI,respectively. In scBoNTAS CGS-C the entire loop is deleted and thedisulfide bridge forming cysteines are replaced by glycine and serine.

B SDS-PAGE analysis of time-dependent cleavage of scBoNT/A wild-type andmutants.

C scBoNTAS wild-type is activated by nBH in a time dependent manner intolight chain and heavy chain within 120 min. Lack of lysines andinsertion of a single arginine residue prolongs the cleavage of the loop(scBoNTAS Throm). A loop lacking any basic residue is still cleavable(scBoNTAS Res). Shortening the loop to 8mer peptide, introducing fiveamino acids with bulky side chains or deleting the entire loop yields anuncleavable scBoNT/A.

FIG. 8: MS/MS analysis of the 50 kDa and 100 kDa cleavage products upondigestion of scBoNT/A with nBH.

A Analysis of the 50 kDa cleavage product which was identified as lightchain of BoNT/A with a Mascot score of 1460. The most C-terminalascribed peptide covers amino acids G433 to K438 which corresponds tothe physiologically observed C-terminus of BoNT/A LC.

B Analysis of the 100 kDa cleavage product which was identified as heavychain of BoNT/A with a Mascot score of 96. The most N-terminal ascribedpeptide covers amino acids A449 to K456 which corresponds to thephysiologically observed N-terminus of BoNT/A HC.

FIG. 9: A The protein content (mg/ml) of anti-nBH-IgY of threesubsequent pools was analysed by 12.5% SDS-PAGE. B ELISA: Nunc MaxisorpF96 microtiter plates were coated with nBH of various lots (500 ng/mL)in PBS overnight at 4° C. and then blocked for 1 h with blocking bufferof PBS containing 0.1% Tween-20 and 2% nonfat skimmed milk. Afterwashing, an IgY dilution of each pool (10 μg/ml in blocking buffer) wasadded for 1 h and detected using biotin-labelled donkey anti-chickenIgY, streptavidin-horseradish peroxidase (both Dianova, Hamburg,Germany) and 3,3′,5,5′-tetramethylbenzidine (Sigma).

FIG. 10: A Recombinant expression and isolation of inactive BH 1-581 (63kDa) by Talon IMAC. 10% SDS-PAGE analysis of Talon IMAC fractions (LMW:116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa; SS34, clear lysate; TD, flowthrough; W, wash fraction; E1-E7, imidazol eluted fractions 1 to 7). BNo endoproteolyis of scBoNT/A into LC (50 kDa) and HC (100 kDa) withrecombinant, iBH (SEQ ID NO: 2; “E”; 63 kDa) is observed at 37° C. after1 h (lane 6)(LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa).

FIG. 11: Use of purified active BoNTHydrolase (nBH) for obtainingproteolytically processed polypeptide

A 200 μg of recombinant purified scBoNT/A is incubated with 350 ngpurified active BoNTHydrolase for 12 min at 37° C. To stop the reactionnBH is removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaPpH 7.5, 150 mM NaCl, sample volume=0.3 ml, flow rate=0.25 ml/min) andthe amount of cleavage is analysed by 10% SDS-PAGE. B Fraction 1 (1800μl) containing ˜40% processed BoNT/A is incubated with 350 ng purifiedactive BoNTHydrolase for 15 min at 37° C. and concentrated to 300 μl byultrafiltration. To finally stop the reaction nBH is removed by SEC(column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl,sample volume=0.3 ml, flow rate=0.25 ml/min) and the amount of cleavageis analysed by 10% SDS-PAGE. C Fractions 1 and 2 (1800 μl) containing˜80% processed BoNT/A are combined and incubated with 120 ng purifiedactive BoNTHydrolase for 25 min at 37° C. and concentrated to 300 μl byultrafiltration. To finally stop the reaction nBH is removed by SEC(column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl,sample volume=0.3 ml, flow rate=0.25 ml/min) and the amount of cleavageis analysed by 10% SDS-PAGE. A >95% processed BoNT/A (SEQ ID NO. 3) isobtained.

THE SEQUENCE LISTING SHOWS

SEQ ID NO: 1: proteolytically active polypeptide derived from aClostridium botulinum strain ATCC 3502, GenBank accession No:“CAL82988.1”, lacking 248 N-terminal amino acid residues

SEQ ID NO: 2: proteolytically inactive polypeptide derived from aClostridium botulinum strain ATCC 3502, GenBank accession No:“CAL82988.1”

SEQ ID NO: 3: BoNT/A of ATCC 3502, Genbank acc. “AAA23262”

SEQ ID NO: 4: Loop of BoNT/A1

SEQ ID NO: 5: Loop of BoNT/A2/A6

SEQ ID NO: 6: Loop of BoNT/A3

SEQ ID NO: 7: Loop of BoNT/A3

SEQ ID NO: 8: Loop of BoNT/A4

SEQ ID NO: 9: Loop of BoNT/A5

SEQ ID NO: 10: Loop of BoNT/A7

SEQ ID NO: 11: Loop of BoNT/B1/B4bv/B6

SEQ ID NO: 12: Loop of BoNT/B2/B3

SEQ ID NO: 13: Loop of BoNT/B5np

SEQ ID NO: 14: Loop of BoNT/C/CD

SEQ ID NO: 15: Loop of BoNT/D

SEQ ID NO: 16: Loop of BoNT/DC

SEQ ID NO: 17: Loop of BoNT/E1-E5

SEQ ID NO: 18: Loop of BoNT/E6

SEQ ID NO: 19: Loop of BoNT/F1/F6

SEQ ID NO: 20: Loop of BoNT/F2/F3

SEQ ID NO: 21: Loop of BoNT/F4

SEQ ID NO: 22: Loop of BoNT/F5

SEQ ID NO: 23: Loop of BoNT/F7

SEQ ID NO: 24: Loop of BoNT/G

SEQ ID NO: 25: Loop of TeNT

SEQ ID NO: 26: nucleic acid sequence encoding SEQ ID NO: 1

SEQ ID NO: 27: nucleic acid sequence encoding SEQ ID NO: 2

The following Examples illustrate the invention and shall, whatsoever,not be construed to limit its scope.

EXAMPLES Example 1: Purification and Characterisation of the NativeBoNTHydrolase (nBH), which Specifically Cleaves Single Chain BoNT/a intoits Active Di-Chain Form

(1) Read-Out System/Activity Test:

To specifically detect and purify an enzymatic activity hydrolysingbotulinum neurotoxin A (BoNT/A) into the 50 kDa light chain (LC) and 100kDa heavy chain (HC) in culture supernatants of C. botulinum and inbetween chromatographic steps we expressed the 150 kDa BoNT/A as singlechain (sc) polypeptide in E. coli. Incubation of the recombinantscBoNT/A with the appropriate enzymatic activity (nBH) should yield a 50kDa LC and a 100 kDa HC visualised by reducing 10-13% SDS-PAGE.

(2) Clostridial Protease Expression:

A single colony of C. botulinum strain ATCC 3502 was inoculated in 100ml brain heart infusion (BHI) media and the culture was incubated overnight at 37° C. under anaerobic conditions. 10 ml of 0/N culture wasinoculated into 1 l BHI media and anaerobically incubated for 48-72 h.

(3) Ammonium Sulphate Precipitation:

The 11 culture supernatant was harvested by centrifugation (4° C.,6500×g, 25 min). Ammonium sulphate was added to a final concentration of85% (here 575 g), the suspension was stirred for 6 hours at 4° C. andsubsequently centrifuged (4° C., 6500×g, 30 min). The pelleted ammoniumsulphate precipitate was dissolved in a small volume (here 5 ml) of 50mM NaP pH 7.5 and dialysed against 50 mM NaP, 150 mM NaCl pH 7.5.Finally, the dialysate was centrifuged (4° C., 40000×g, 60 min) and thesupernatant used for the IEC.

(4) Ion Exchange Chromatography (IEC, Column HiPrep 16/10 Q FF):

Supernatant of (3) (FIG. 1, lane 3) was applied to a HiPrep 16/10 Q FFanion-exchange column equilibrated and run with a buffer containing 50mM NaP pH 7.5, 150 mM NaCl. The run was performed at a flow rate of 1ml/min. An activity test was performed by incubating 5 μl of every otherfraction with 2 μg scBoNTA for 1 h at 37° C. and subsequent analysis onSDS-PAGE (FIG. 1). Fractions 6-24 were combined and its volumeconcentrated to 3.5 ml by using ultrafiltration (Amicon-Ultra MWCO10,000).

(5) Size Exclusion Chromatography (SEC, HiLoad 16/60 Superdex 200):

Subsequently, the concentrated protein solution of (4) was loaded on aHiLoad 16/60 Superdex 200 column, equilibrated with 50 mM NaP pH 7.5,150 mM NaCl. Separation was performed at a flow rate of 1 ml/min.Fractions with a retention volume between 80 ml and 100 ml were analysedusing the activity test (1) and the appropriate fractions containing theenzymatic activity (nBH) were combined (˜10 ml) and concentrated to 3 mlby ultrafiltration. Subsequently, ammonium sulphate was added to a finalconcentration of 12.5%=500 mM (+0.2 g).

(6) Hydrophobic Interaction Chromatography (HIC, HiTrap PhenylSepharose):

nBH was bound to the Phenyl Sepharose in buffer A (50 mM NaP pH 7.5, 500mM ammonium sulphate). Bound nBH was eluted by reducing the amount ofammonium sulphate due to a linear increasing gradient with buffer B (50mM NaP pH 7.5) at a flow rate of 1 ml/min. All protein containingfractions were analysed using the activity test (1) and the appropriatefractions were combined and concentrated by ultrafiltration to 3.5 ml.The buffer of the solution was adjusted to 50 mM NaP pH 7.5; 150 mMNaCl.

(7) SEC (HiLoad 16/60 Superdex 75):

Finally, the nBH was purified by SEC using the HiLoad 16/60 Superdex 75column at 50 mM NaP pH 7.5, 150 mM NaCl and a flow rate of 1 ml/min.Fractions with a retention volume between 70 ml and 80 ml were analysedby 12.5% SDS-PAGE (FIG. 2) and the fractions 8-12 containing the nBHwhich migrates at ˜37.3 kDa were combined (˜10 ml) and concentrated to 1ml by ultrafiltration.

(8) The prominent protein migrating at approximately 37.3 kDa (nBH) wasanalysed by N-terminal peptide sequencing according to Edman degradationprotocol. The sequence of the identified peptide is V Q G Q S V K G V Gand corresponds to the first ten residues of SEQ ID NO: 1.(9) Two lots of nBH (NT02CB1447, 37.3 kDa, FIG. 3, lane 3: TIK301009,lane 4: TIK280509) were reproducibly isolated according to the procedureas described above. Following modifications of the isolation procedureyields the nBH isoform NT02CB1446 (38.6 kDa, FIG. 3, lane 2, lotTE311206): (i) growth of C. botulinum culture: 18 h instead of 48 to 72h; (ii) variation of chromatographic steps: IEC→SEC Superdex 75→HICPhenyl Sepharose instead of IEC→SEC Superdex 200→HIC PhenylSepharose→SEC Superdex 75.

Example 2: Sequence Identification of nBH from C. botulinum by MassSpectrometry (MS)

(1) Tryptic Digestion:

The protein bands migrating at approximately 38 kDa (nBH) in SDS-PAGEwere excised for tryptic digestion and destained by gently shaking in 50mM NH₄HCO₃, 50% acetonitrile for 30 min at 37° C. Destaining wasrepeated until gel spots were clear. Acetonitrile (100%) was added andremoved after 3 min. Subsequently, spots were dried in a speed vacsystem (Eppendorf, Germany). Trypsin (10 ng/μl) in 50 mM NH₄HCO₃ wasadded and incubated on ice for 1 h. Then, the remaining trypsin solutionwas removed, a small volume 50 mM NH₄HCO₃ was added and digestion wascarried out at 37° C. over night. The supernatant was collected and gelpieces were extracted using 5% TFA, 10% acetonitrile for two times. Allfluids were combined, dried in a speed vac and extracted peptides werestored at 4° C.

(2) Matrix Assisted Laser Desorption Ionisation Time of Flight(MALDI-TOF/TOF) MS:

Samples were analyzed in an MALDI-TOF/TOF mass spectrometer (Ultraflex1Bruker Daltonik GmbH) in linear mode with an acceleration voltage of 25kV. Masses were detected from 700 m/z to 4,500 m/z. Samples (2 μl) werecocrystallised with 2 μl of sinnapinic acid solution containing 50%acetonitril and 0.2% trifluoric acetic acid (TFA) directly on astainless steel MALDI target plate. 500 laser shots were collected foreach sample.

(3) Peptide Separation by Reversed Phase Chromatography:

Peptide separation was done by reversed phase chromatography using anano-HPLC system (Agilent Technologies, Waldbronn, Germany) that consistof an autosampler and a gradient pump. The sample was dissolved inbuffer A (5% acetonitril, 0.1% formic acid) and an aliquot of up to 10μl was injected onto a C18 column (Zorbax SB-C18, 5 μm, 300 A, 0.5 mminner diameter, length 15 cm) at a flow rate of 5 μl/min. After loading,the column was washed for 15 min with buffer A and the peptides wereeluted using a gradient of eluent A and eluent B (70% (v/v) acetonitrilein 0.1% (v/v) formic acid) from 0% to 100% eluent B in 75 min.

(4) Electrospray Ionisation (ESI)-Interface and Ion Trap MassSpectrometry:

The HPLC outlet was directly connected to the nanoESI source of an iontrap mass spectrometer and the Agilent coaxial sheath-liquid sprayer wasused (Agilent Technologies). The outlet capillary was hold by asurrounding steel needle and looked 0.1 to 0.2 mm out of it. The spraywas stabilized by N₂ as nebulizer gas (5 l/min). Ionization voltage wasset to 4,500 V and dry gas was applied at 5 psi and 250° C. Spectra werecollected with an Esquire3000+ ion trap mass spectrometer (BrukerDaltonik) at a scan speed of 13,000 m/z per second. Using ESI inpositive mode, mass spectra were acquired from 50 to 1600 m/z inscanning mode and data dependent switching between MS and MS/MSanalysis. To increase the quality of MS/MS spectra only two precursorions from one spectrum were selected for MS/MS analysis and activeexclusion was set to 2 min to exclude precursor ions that had alreadybeen measured.

(4) Data Processing:

Data processing was performed with the Data Analysis (version 3.0) andBioTools (version 3.0) software packages (Bruker Daltonik). Proteinidentification was done using MASCOT software (version 2.1) and MSDBdata base (Matrix Science, London, UK).

(5) Results:

TABLE 2 nBH idntified by MS Protein Genbank aa of MW Mascot lane Lot nBHconcentr. Name of ORF acc. ORF [kDa] score 2 TE311206 192 ng/μlNT02CB1446 CAL82987.1 254-594 38.6 725 CBO1444 3 TIK301009 130 ng/μlNT02CB1447 CAL82988.1 249-581 37.3 555 CBO1445 4 TIK280509 114 ng/μlNT02CB1447 CAL82988.1 249-581 37.3 609 CBO1445

The 38.6 kDa protein band of lane 2 (nBH lot TE311206) was identified asNT02CB1446/CBO1444 with a Mascot score of 725 and a peptide MS/MSsequence coverage of 29.6% over the entire open reading frame (ORF). Nopeptide was identified derived from the N-terminal 253 amino acids (FIG.4). The MS/MS analysis of lot TE311206 displayed a sequence coverage of52% according to the C-terminal amino acids 254-594 forming the nBH.

The 37.3 kDa protein bands of lane 3 (nBH lot TIK301009) and lane 4 (nBHlot TIK280509) were identified as NT02CB1447/CBO1445 with a Mascot scoreof 555 and 609, respectively. Except one all peptides identified derivefrom the C-terminal 333 amino acids (FIG. 5). The MS/MS analysis of lotTIK301009 displayed a sequence coverage of 49.5% according to theC-terminal amino acids 249-581 forming the nBH.

Example 3: Characterisation of Enzymatic Specificity of nBH

(1) The concentration dependent proteolytic activity of nBH derived fromthree purification batches was compared (FIG. 6). An activity testanalysing nBH derived from the batches TIK301009, TIK280509 and TE311206using various dilutions of nBH demonstrates that higher dilutionsdecrease the cleavage rate. The proteolytic activity of the threedifferent batches is nearly identical indicating that the maturatedisoform NT02CB1446 (TE311206) displays a similar specific activity asthe maturated NT02CB1447 (SEQ ID NO: 1).(2) The time-dependent cleavage of scBoNT/A wild-type and mutants by nBHwas analysed employing the activity test (FIG. 7). scBoNTAS wild-type isactivated by nBH in a time dependent manner into light chain and heavychain within 120 min by more than 95%. The loop sequence was modified tocharacterise the cleavage site. In scBoNTAS Throm all lysine residuesare removed and the thrombin recognition sequence LVPRGS is insertedwhich prolonged the cleavage rate. In scBoNT Res the loop lacks anybasic amino acids which drastically delays the complete hydrolysisindicating a strong recognition preference of nBH for basic residueslike lysine and arginine at the cleavage site. Furthermore,accessibility of nBH to the loop by is impaired by shortening the loopto eight small residues or five amino acids with bulky side chains(scBoNTAS (GGSG)₂ and scBoNTAS FQWYI).(3) The MS/MS analysis of the 50 kDa cleavage product upon digestion ofscBoNT/A with nBH exhibited that the most C-terminal ascribed peptidecovers amino acids G433 to K438 which corresponds to the physiologicallyobserved C-terminus of BoNT/A LC (FIG. 8A). Analysis of the 100 kDacleavage product which was identified as heavy chain of BoNT/Ademonstrated that the most N-terminal ascribed peptide covers aminoacids A449 to K456 which corresponds to the physiologically observedN-terminus of BoNT/A HC (FIG. 8B). Thus, the isolated nBH yieldsphysiologically processed BoNT/A and preferentially hydrolyses peptidebonds C-terminal to lysine and arginine residues.

Example 4: Evolutionary Conservation of BoNTHydrolase and its Isoforms

Protein sequence analysis of SEQ ID NO: 2 (Genbank acc.CAL82988.1/YP_001253958.1) revealed three conserved domains. Residues18-573 correspond to a Zinc metalloprotease (elastase) or LasB involvedin amino acid transport and metabolism with a Blast score of 738.Residues 148-212 correspond to a peptidase propeptide and YPEB domain orPepSY (Blast score 97. Residues 336-573 are part of the peptidase M4family including thermolysin, protealysin, aureolysin and neutralproteases (Blast score 803).

The genome sequencing of C. botulinum ATCC 3502 has revealed theexistence of six ORFs encoding iBH isoforms (Sebaihia et al., 2007,Genome Res. 17(7):1082-1092). Further genome data is available for 10group I C. botulinum strains as well as the non-BoNT secreting C.sporogenes which all contain between five to seven ORFs encoding iBH.The nBH (SEQ ID NO: 1) shares minimum 64% amino acid sequence identitywith the other 63 isoforms.

Example 5: Generation of Antibodies Specific for the BoNTHydrolase

(1) Generation of IgY:

Sixteen-week-old chickens [ISA Brown and Lohmann Selected Leghorn (LSL),Spreenhagener Vermehrungsbetrieb für Legehennen GmbH, Bestensee,Germany] were kept in individual cages, exclusively constructed for themaintenance of chickens (Ebeco, Castrop-Rauxel, Germany). Food (ssniffLegehühner-Zucht 1 and 2; ssniff Spezialitäten GmbH, Soest, Germany) andwater were available ad libitum, and the chickens started laying eggsbetween 23 and 25 weeks of age. Eggs were collected daily, labelled, andstored at 4° C. until they were further processed. All animalmaintenance and experiments were performed in accordance with theguidelines of local authorities, Berlin (No. H0069/03). Chickens wereimmunized and boosted via the i.m. route (pectoralis muscle, left andright side) a total of 10 times over a 1-year period, with intervalsbetween 4 and 8 weeks. The interval used was based on previous work thatshowed no demonstrable memory cells until at least 3 weekspostimmunisation (Pei and Collisson, 2005). The antigen concentrationused was approximately 20 μg per injection (nBH). No more than 500 μl ofantigen solution was injected per immunization. Freund's completeadjuvant was used for the first immunisation, and FIA was used for thesubsequent booster injections. The method for IgY purification wasadapted from Polson et al. (1980). Briefly, the egg yolk was diluted 1:2with sterile PBS (pH 7.4, Roche, Mannheim, Germany). For elimination oflipids and lipoprotein, 3.5% (w/v) polyethylene glycol (PEG) 6000 (Roth,Karlsruhe, Germany) was added. After gentle shaking followed bycentrifugation (10,000×g for 20 min at 4° C.), the supernatant wasdecanted and solid PEG 6000 was added to a final concentration of 12%(w/v). This mixture was then centrifuged as above. The precipitate wasdissolved in 10 ml of PBS, PEG was added to 12% (wt/vol), and thesolution was centrifuged. Finally, the precipitate was dissolved in 1.2ml of PBS, transferred into a microdialysis device (QuixSep, Roth,Germany) and dialysed against PBS at 4° C. The protein content (mg/ml)was analysed by 12.5% SDS-PAGE (FIG. 9A) and measured photometrically at280 nm and was calculated according to the Lambert-Beer law with anextinction coefficient of 1.33 for IgY.

(2) ELISA:

Nunc Maxisorp F96 microtiter plates (VWR International GmbH, Darmstadt,Germany) were coated with nBH of various lots (500 ng/mL) in PBSovernight at 4° C. and then blocked for 1 h with blocking buffer of PBScontaining 0.1% Tween-20 and 2% nonfat skimmed milk (Merck, Darmstadt,Germany). After washing, a IgY dilution (10 μg/ml in blocking buffer)was added for 1 h and detected using biotin-labeled donkey antichickenIgY, streptavidin-horseradish peroxidase (both Dianova, Hamburg,Germany) and 3,3′,5,5′-tetramethylbenzidine (Sigma). Detected nBH isillustrated in FIG. 9B.

(3) Western Blot:

nBH was separated per 12.5% SDS-PAGE, and were transferred onto apolyvinylidene fluoride membrane (Invitrogen GmbH, Karlsruhe, Germany)using standard immunoblotting techniques. The membrane was blockedovernight at 4° C., and incubated with IgY (1:5,000 in blocking buffer)for 1 h. After washing, the membrane was probed with biotin-labelleddonkey anti-chicken IgY for 30 min and was developed using alkalinephosphatase and CDP-Star (Perkin Elmer, Waltham, Mass.).

Example 6: Recombinant Expression of BoNTHydrolase

(1) Plasmid Constructions:

The gene portions encoding native BH (SEQ ID NO: 1) and its propeptide(SEQ ID NO: 2) were amplified by PCR using suitable oligonucleotides andgenomic DNA of C. botulinum ATCC 3502, fused to an oligonucleotidecoding for the His6Tag and inserted into pQE3 (Qiagen) yielding theexpression plasmid pQ-BH1445H6-249-581 and pQ-BH1445H6-1-581,respectively. Nucleotide sequences were verified by DNA sequencing.

(2) Purification of Recombinant Proteins:

nBH and iBH, fused to a carboxyl-terminal His6Tag, were producedutilizing the E. coli strain M15pREP4 (Qiagen) during ten hours ofincubation at room temperature, and were purified on Talon-sepharosebeads (Clontech Inc.) following to the manufacturer's instructions.Fractions containing the desired proteins were pooled, frozen in liquidnitrogen, and kept at −70° C. iBH was isolated as recombinant proteinwith a MW of 63 kDa (FIG. 10A). The inactivity of iBH was demonstratedusing the activity test: after 1 h at 37° C. no scBoNT/A wt washydrolysed in LC and HC (FIG. 10B).

Example 7: Inhibition of BoNTHydrolase

(1) Screening for peptide inhibitors of BH: Peptides based on SEQ IDNOs: 4 to 25 will be synthesised lacking one or more basic residues.Each peptide will be added to the mixture according to the activitytest. A peptide being able to decrease the amount of processed scBoNT/A,prolong the duration required for fully processing scBoNT/A or blockprocessing scBoNT/A is considered to be an inhibitor of nBH.(2) Screening for antibody-based inhibitors: Antibodies generatedagainst epitopes derived from nBH like IgY of Example 5 are incubatedwith nBH and subsequently subjected to the activity test. An antibodybeing able to decrease the amount of processed scBoNT/A, prolong theduration required for fully processing scBoNT/A or block processingscBoNT/A is considered to be an inhibitor of nBH.

Example 8: Use of Purified Active BoNTHydrolase (nBH) for ObtainingProteolytically Processed Polypeptide

(1) 200 μg of recombinant purified scBoNT/A is incubated with 350 ngpurified active BoNTHydrolase for 12 min at 37° C. To stop the reactionnBH is removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaPpH 7.5, 150 mM NaCl, sample volume=0.3 ml, flow rate=0.25 ml/min) andthe amount of cleavage is analysed by 10% SDS-PAGE (FIG. 11A).(2) Fraction 1 (1800 μl) containing ˜40% processed BoNT/A is incubatedwith 350 ng purified active BoNTHydrolase for 15 min at 37° C. andconcentrated to 300 μl by ultrafiltration. To finally stop the reactionnBH is removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaPpH 7.5, 150 mM NaCl, sample volume=0.3 ml, flow rate=0.25 ml/min) andthe amount of cleavage is analysed by 10% SDS-PAGE (FIG. 11B).(2) Fractions 1 and 2 (1800 μl) containing ˜80% processed BoNT/A arecombined and incubated with 120 ng purified active BoNTHydrolase for 25min at 37° C. and concentrated to 300 μl by ultrafiltration. To finallystop the reaction nBH is removed by SEC (column Superdex 200 10/300 GL,buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume=0.3 ml, flowrate=0.25 ml/min) and the amount of cleavage is analysed by 10% SDS-PAGE(FIG. 11C). A >95% processed BoNT/A (Seq ID NO. 3) is obtained. Theidentical fully processed second polypeptide (>95% processed BoNT/A) isobtained if the second polypeptide is processed in one step for 50 minat 37° C. (200 μg scBoNT/A incubated with 350 ng nBH). After anincubation time of 1 h at 37° C., more than 97% of BoNT/A is processed.

The invention claimed is:
 1. A method for manufacturing a di-chainbotulinum neurotoxin serotype A (BoNT/A) or derivative thereof, themethod comprising contacting a Lys-C polypeptide with a single-chainBoNT/A or derivative thereof; wherein the single-chain BoNT/A orderivative thereof is proteolytically processed by the Lys-C polypeptideto produce the di-chain BoNT/A or derivative thereof.
 2. The method ofclaim 1, wherein the single-chain BoNT/A or derivative thereof is anaturally-occurring neurotoxin, a recombinant neurotoxin, a modifiedneurotoxin, a neurotoxin lacking the native H_(C) domain or partsthereof, or a BoNT/A derivative with other amino acid residues replacingthe neurotoxin H_(C) domain.
 3. The method of claim 2, wherein thesingle-chain BoNT/A or derivative thereof comprises an amino acidsequence having at least 50% sequence identity with a polypeptidesequence selected from any one of SEQ ID NOs: 3 to
 10. 4. The method ofclaim 1, wherein the C-terminus of the L-chain and the N-terminus of theH-chain of the di-chain BoNT/A or derivative thereof are identical tothe corresponding C-terminus of the L-chain and the N-terminus of theH-chain of di-chain BoNT/A isolated from wild-type clostridia.
 5. Themethod of claim 1, wherein the di-chain BoNT/A or derivative thereof hasan identical amino acid sequence as the corresponding di-chain BoNT/Agenerated from the same single-chain BoNT/A polypeptide in wild-typeclostridia.
 6. The method of claim 1, wherein the step of contacting theLys-C polypeptide and the single-chain BoNT/A or derivative thereofoccurs within a cell, in a cell lysate, in a purified cell lysate, or ina subject.
 7. The method of claim 3, wherein the Lys-C polypeptideproteolytically cleaves the single-chain BoNT/A or derivative thereof ata position immediately C-terminal to a basic amino acid residue withinthe sequence of any one of SEQ ID NOs: 3 to 10.